Basc02 Full Dna

The Structure and Function
of Nucleic Acids
Revised edition
C.F.A. Bryce* and D. Pacini
*Department of Biological Sciences, Napier University, Edinburgh, and
Bolton School Boys Division, Bolton

The Biochemistry Across the School Curriculum Group (BASC) was set
up by the Bi ochemi cal Soci ety i n 1985. I ts membershi p i ncl udes edu-
cati on professi onal s as wel l as Soci ety members wi th an i nterest i n
school science education. I ts first task has been to produce this series of
booklets, designed to help teachers of syllabuses which have a high bio-
chemical content.
Other topi cs covered by thi s seri es i ncl ude: Essential Chemistry for
Biochemistry; Enzymes and their Role in Biotechnology; Metabolism;
I mmunology; Photosynthesis; Recombinant DNA Technology; Biological
Membranes; and The Biochemical Basis of Disease.
More information on the work of BASC and these booklets is available
from the Educati on Offi cer at the Bi ochemi cal Soci ety, 59 Portl and
Place, London W1N 3AJ.
Comments on the content of this booklet will be welcomed by the Series
Editor Mrs D. Gull at the above address.
ISBN 0 90449 834 4
The Biochemical Society 1998
First edition published 1991
Al l BASC materi al i s copyri ght by the Bi ochemi cal Soci ety. Extracts
may be photocopied for classroom work, but complete reproduction of
the enti re text or i ncorporati on of any of the materi al wi th other doc-
uments or coursework requires approval by the Biochemical Society.
Printed by Holbrooks Printers Ltd, Portsmouth, U.K.
iii
Contents
Foreword................................................................................v
1
Nucleic acids and genetic information transfer ................1
Learning objectives.................................................................................1
Introduction.............................................................................................1
2
Isolation and structure of nucleic acids..............................5
Learning objectives.................................................................................5
Sugars........................................................................................................6
Bases.........................................................................................................7
Inorganic phosphate...............................................................................8
Building nucleic acids from their building blocks..............................8
Nucleoside di- and triphosphates.......................................................9
Nucleic acids.........................................................................................10
DNA isolation,base composition and structure...........................12
RNA isolation,base composition and structure............................17
3
DNA replication.................................................................21
Learning objectives..............................................................................21
Introduction..........................................................................................21
The Meselson and Stahl experiment................................................23
Mechanism of replication....................................................................25
4
The genetic code................................................................29
Introduction..........................................................................................29
How many bases code for an amino acid?......................................31
Evidence for a triplet code.................................................................32
Deciphering the code..........................................................................33
Nonsense codons................................................................................34
iv
5
Transcription......................................................................37
Introduction..........................................................................................37
Mechanism of transcription...............................................................38
6
Translation..........................................................................41
Introduction..........................................................................................41
Activation of tRNA..............................................................................42
Initiation.................................................................................................43
Elongation..............................................................................................44
Termination...........................................................................................45
Polysomes..............................................................................................46
7
Assessment:past examination questions and outline
solutions..............................................................................47
Questions..............................................................................................47
Answers.................................................................................................56
8
Laboratory practicals........................................................59
Extraction and characterization of DNA from Micrococcus
lysodeikticus............................................................................................59
Purification of RNA.............................................................................61
Further practicals.................................................................................62
9
Source materials................................................................63
Textbooks..............................................................................................63
Articles...................................................................................................63
Teaching aids..........................................................................................64
Subject index......................................................................65
v
Foreword
A study of the structure and functi on of nucl ei c aci ds i s needed to be
able to understand how information controlling the characteristics of an
organism is stored in the form of genes in a cell and how these genes are
transmitted to future generations of offspring. The rapid developments
i n the area of geneti c engi neeri ng and recombi nant DN A technol ogy
(whi ch are covered i n Bookl et 7 of the BASC seri es) have onl y been
possi bl e as a resul t of detai l ed understandi ng of the structure of DNA
and RNA. I t i s therefore not surpri si ng to see nucl ei c aci ds i ncl uded i n
the compul sory core subject matter of al l of the l i near and modul ar A-
level Biology syllabuses. The major purpose of these guidance notes is to
provide an account of the role of DNA and RNA in these processes in at
l east suffi ci ent detai l for A-l evel study. Addi ti onal i nformati on more
sui ted for general i nterest, Oxbri dge preparati on or fi rst-year under-
graduate study is placed in shaded boxes of text.
I n order to maximize the benefit of this booklet for A-level students
and thei r teachers, a set of l earni ng objecti ves have been i ncl uded at the
start of chapters 16. These objecti ves are based upon the rel evant
knowl edge and understandi ng statements of current A-l evel Bi ol ogy
syl l abuses. Chapter 7 consi sts of recent A-l evel exami nati on questi ons
and answers and this should help students get a feel for the level of detail
required. We are grateful to the various Examinations Boards, especially
the Northern Exami nati ons and Assessment Board (NEAB), for thei r
permission to include this material.
1
Nucleic acids and genetic
information transfer
Learning objectives
Each student should, without reference to his or her notes, be able to:
state that the genoty pe descri bes the geneti c composi ti on of an
organism in terms of specific genes, i.e. alleles that it contains;
state that the phenotype i s the col l ecti on of vi si bl e characteri sti cs
which identify an individual organism;
state that the phenotype of an i ndi vi dual depends not onl y on i ts
genotype but also on the environment in which it lives; and
state that DNA, which is found in the nucleus of a cell, contains and
i s responsi bl e for the transfer of geneti c i nformati on, i .e. the
genotype.
Introduction
N ormal l y, when we tal k about genes and heredi ty we perhaps thi nk
i mmedi atel y of how chi l dren resembl e thei r parents or, dependi ng on
our background knowl edge, we may even thi nk about Mendel and the
hei ght and col our of hi s pea pl ants. Thi s l evel of geneti cs i s known as
phenotypic expression, where the phenotypeof an individual is the col-
l ecti on of vi si bl e and recogni zabl e characteri sti cs by whi ch that i ndi -
vidual is identified.
The phenoty pe i s i nfl uenced by the envi ronment as wel l as the
genotype; but this aspect is more appropriate to classical genetics and will
not concern us at the present time. I n this section we are more concerned
with the collection of the inherited factors which determine these traits,
1
and this is a cells genotypic expression. The genotypeis the genetic com-
position of an organism in terms of the forms of specific genes, i.e. alleles,
that it contains, and its study is referred to as molecular genetics.
The very fact that a chi l d can resembl e one or both parents means
that there was some way i n whi ch the geneti c i nformati on was passed
from the parent to the chi l d. What then i s the nature of the geneti c
materi al ? The answer to thi s questi on came from the experi mental
observations shown in Table 1.
More to heredity than DNA
In similar types of nuclear transplantation experiments,scientists have recently
revealed that the environment of the nucleus (i.e.the cytoplasm in which it finds
itself) can alter the functioning of key genes,including those which determine
body size.These have been referred to as epigeneticchanges,these being
changes which affect the way DNA works rather than sequence changes.These
findings could have implications for in vitrofertilization in humans.
New Scientist (19 April 1997)
Single-cell organisms take a siesta
In 1997 a short report appeared which indicated that a study on the patterns of
DNA synthesis in single-cell organisms revealed that cells synthesized DNA from
sunrise to about noon,then shut down synthesis for between three and six
hours,then restarted before sunset.The explanation given for this phenomenon is
that this represents an evolutionary throwback to the Precambrian times when
there was no ozone layer and hence the suns ultraviolet light at its peak could
have damaged the DNA.This may have a future significance if the ozone layer
does continue to degrade.
New Scientist (4 January 1997)
Such studi es represent the most concl usi ve evi dence that DNA i s
responsi bl e for the transfer of geneti c i nformati on, at l east i n mi cro-
organi sms and vi ruses. The evi dence that thi s i s al so true i n hi gher
organi sms (pl ants and ani mal s), al though somewhat l ess concl usi ve,
leaves no doubt that this is the case. Since some virus particles have been
found that have no DN A, but whi ch have RN A i nstead, i t i s more
correct to say that nucleic acids are the genetic information carriers.
The exact rol es of DN A and RN A i n the compl ex process of the
transfer of genetic information are the subjects of subsequent sections of
this booklet.
2 Nucleic acids:structure and function
N
u
c
l
e
i
c

a
c
i
d
s

a
n
d

g
e
n
e
t
i
c

i
n
f
o
r
m
a
t
i
o
n

t
r
a
n
s
f
e
r
3
Table 1. Steps in the discovery of DNA as the genetic material
Experimenter(s) and dates Details of procedure Conclusion
Late 19th century biologists Removal of the nucleus of the cell produced death, Something in the nucleus is responsible for a
but removal of the same volume of cytoplasm did not. cells survival.
Late 19th century biologists Removal of a cell nucleus followed by transplantation Something in the nucleus is responsible for
of the nucleus from a different cell type changed the the phenotype of a cell.
shape and function of the donor cell.
Griffiths (1929) Dead,disease-producing,virulent strains of Pneumococcus Something had been transferred from the
were able to transform harmless cells of non-virulent dead organisms that altered the phenotype of
Pneumococcusinto lethal organisms. the living cells.
Avery and MacLeod (1944) These workers extracted the DNA from the virulent DNA is responsible for cell transformation
Pneumococcusbacterium.On adding it to non-virulent i.e.a cells phenotype.
strains these were transformed into disease-causing
organisms.The DNA contained no detectable protein
and was unaffected by proteases,but the transformation
was affected by use of DNase.
Hershey and Chase (1953) These workers infected Escherichia coli bacteria using a DNA is responsible for cell transformation
radioactive strain of T2 bacteriophage virus in which the i.e.a cells phenotype.
phosphorus atoms in the DNA and the sulphur atoms in
the coat protein were labelled.This revealed that only the
DNA entered the bacteria,causing transformation.
2
Isolation and structure of
nucleic acids
Learning objectives
state that DNA is composed of phosphate, deoxyribose and the four
major bases: adenine, guanine, cytosine and thymine;
state that RNA is composed of phosphate, ribose, adenine, guanine,
cytosine and uracil;
state that DNA and RNA are polymers of nucleotide subunits;
state that a nucleotide is composed of a phosphate group, a pentose
sugar and one of the four corresponding bases;
state that the backbone of a DNA mol ecul e i s a chai n of repeati ng
deoxyribosephosphate units;
state that the backbone of an RNA mol ecul e i s a chai n of repeati ng
ribosephosphate units;
state that each molecule of DNA is usually composed of two chains;
state that in DNA, adenine will only bind with thymine on opposite
chains and guanine will only bind with cytosine on opposite chains;
state that the two linked chains in DNA are arranged in a double helix;
state that there are three pri nci pal ty pes of RN A, these bei ng
messenger RN A (mRN A), transfer RN A (tRN A) and ri bosomal
RNA (rRNA), which are all single-stranded molecules;
state the function of the three principal types of RNA;
draw a di agram of a si mpl e l adder-l i ke representati on of a DN A
molecule; and
state that DN A i s a stabl e pol y nucl eoti de whi ch contai ns coded
genetic information for inherited characteristics.
5
Introduction
Al though the nucl ei c aci ds were fi rst di scovered i n 1868, by Fri edri ch
Mi escher worki ng wi th pus cel l s obtai ned from di scarded surgi cal
bandages, i t was not real l y unti l the earl y 1940s that the chemi stry and
biology of the nucleic acids were set on firm foundations.
Basi cal l y, nucl ei c aci ds can be subdi vi ded i nto two types: deoxy-
ribonucleic acid (DNA)and ribonucleic acid (RNA). Both DNA and
RNA have been shown to consist of three groups of molecules: pentose
(5-carbon-atom) sugars; organic bases; and inorganic phosphate.
Sugars
There are only two types of sugar present in nucleic acids, ribosewhich
i s present sol el y i n RN A (hence i ts name) and deoxyribosewhi ch i s
present sol el y i n DNA (agai n, the sugar gi ves ri se to the name deoxy-
ri bonucl ei c aci d). The chemi cal structures for these compounds are
shown here:
The prefi x deoxy means without oxygen, and we can see from
the structures that the only difference between them is the absence of an
oxygen in the deoxyribose sugar (see shaded area). Both sugars contain 5
carbon atoms (pentose sugars) and for convenience we number these as
shown in the next figure. The dash or prime () on, for example, the 5
i ndi cates the carbon i n the ri bose ri ng. The puri ne or pyri mi di ne ri ngs
(see Bases, below) are numbered without primes in order to distinguish
them.
6 Isolation and structure of nucleic acids
Ribose Deoxyribose
OH OH OH
OH OH
No OH
group
O
H
O
HOCH
2
HOCH
2
Bases
The nucl eoti de bases found i n nucl ei c aci ds are rel ated ei ther to the
purinering system or to the pyrimidinering system.
I n DN A we fi nd pri nci pal l y four di fferent bases: adenine (A),
guanine (G), cytosine (C) and thymine (T). The fi rst two are deri ved
from purine whereas the remaining two are derived from pyrimidine. I n
RN A we fi nd pri nci pal l y four di fferent bases; adeni ne, guani ne and
cytosi ne as i n DNA. The fourth base i n RNA, however, i s not thymi ne
but instead the pyrimidine-derived base, uracil (U).
The chemi cal structures for each of the fi ve bases are shown bel ow.
Nucleic acids:structure and function 7
OH OH
OH
C
H
C C
H
C
O
H H
HOCH
2
5
4 1
2 3
Purine Pyrimidine
N N
N
N
H
N
N
Purines
Adenine Guanine
Cytosine Uracil Thymine
Pyrimidines
N HN
HN
H
2
N
HN
CH
3
N
NH
2
NH
2
H
N N
H
N N
N
N
H
N
H
N
H
O O O
O
O
C
N
C
O
Note: A-level students are not expected to recognize these structural
formulae. For further help in understanding the chemistry of these com-
pounds, refer to BASC Booklet 1, Essential Chemistry for Biochemistry.
I n addi ti on to these major bases there i s al so a l arge range of so-
cal l ed minor baseswhi ch occur l ess frequentl y than the others, e.g. 5-
methyl cytosine.
Inorganic phosphate
There are phosphate resi dues i n nucl ei c aci ds and they are of the type
derived from phosphoric acid, the structure of which is shown below.
Building nucleic acids from their building blocks
When any one of the bases i s joi ned to ei ther one of the two sugar mol -
ecules, we have a compound known as a nucleoside. I f the sugar residue
is ribose then we have a ribonucleoside, whereas if it is deoxyribose then
we have a deoxyribonucleoside. The bond l i nki ng these structures i s
known as a glycoside bond.
Addi ti on of a phosphate group to the sugar resi due of a nucl eosi de
molecule produces a different molecule called a nucleotide. Examples of
various nucleotides are shown below.
OH
OH
O
P O H
Deoxycytidine 5-monophosphate Uridine 5-monophosphate
HO P
O
OH
OH
2
C HO P
O
OH
OH
2
C
HN
OH
NH
2
H OH H OH H H
N N
N
O
O
O
O
O
N ucl eoti des can be regarded as the bui l di ng bl ocks for the l arger
nucleic acid molecules, DNA and RNA.
Nucleoside di- and triphosphates
One or two addi ti onal phosphates can be added to the fi rst phosphate
group of a nucleoside molecule (a nucleoside monophosphate, NMP) by
means of a pyrophosphate linkage. The mol ecul es formed i n thi s way
are called nucleoside diphosphates (NDPs) and nucleoside triphosphates
(N TPs). The most i mportant base i nvol ved i n these compounds i s
adenine, forming the adenosine mono-, di- and triphosphate molecules
(AMP, ADP and ATP), which fulfil vital roles in many cellular processes
as you may know from your study of respi rati on and photosynthesi s
[for further detai l s see BASC Bookl ets 4 (Metabolism) and 6 (Photo-
synthesis)].
OH OH OH
OH OH
O O O

O O O O
Base
NMP
NDP
NTP
H H
H H
CH
2
P P P O H
Nucleic acids
Nucl ei c aci ds are formed by the combi nati on of nucl eoti de mol ecul es
through sugarphosphate bonds known as phosphodiester linkages.
Because a nucleic acid is a polymer of many nucleotide molecules, DNA
and RNA molecules are called polynucleotides.
The structure of a polynucleotide is shown diagrammatically above.
I n common with the formation of other biological polymers, e.g. starch
and proteins, water molecules are produced by condensationreactions
and energy from ATP mol ecul es, produced i n cel l ul ar respi rati on, i s
used.
35
phosphodiester
linkage
35
phosphodiester
linkage
35
phosphodiester
linkage
3 end
5 end
Adenine (A)
Guanine (G)
Thymine (T)
Cytosine (C)
O
O
P
O
H H
N
N
N
NH
2
H
3
C
NH
2
NH
2
NH
OH
NH
CH
2
CH
2
CH
2
CH
2
N
N
N
N
N
N
H
H
H
H H
H
H
H
H H
N
H
H
H
H H
H
H
H
O O
O
O
P
O
O
O
O
P
O
O O O
O
O
P
O
O O
O O
O
O
5
4
3 2
5
4
3 2
5
4
3 2
5
4
3 2
From thi s fi gure i t can be seen that the phosphodi ester bond i s
formed between the 5carbon of one sugar molecule and the 3 carbon of
the next. The doubl e-stranded nature of DNA i s shown schemati cal l y
above.
Si nce i t woul d be very ti me consumi ng to have to wri te out such a
structure every ti me i t was needed, abbrevi ated forms have been
introduced. These are essentially of three types:
I f the P i s on the l eft of a base then i t i s attached to the 5-carbon
(five); if it is on the right then it is attached to the 3-carbon (three).
Sugar
Phosphate
group
Phosphodiester
bond
A T
C G
G C
T A
ApGpCpGpUp
5
AGCGU
3
DNA isolation,base composition and structure
Isolation
I n order to study the chemistry of DNA it must be isolated and purified
from the nucleus of a cell. I n the isolation of DNA from cell samples, the
major contami nant i s histone proteinwhi ch i s cl osel y associ ated wi th
DNA to form visible chromatinthreads in the nucleus. To remove this
contaminant the cell is first deproteinized by treatment with phenol and
the DNA is precipitated using ice-cold ethanol. A more detailed account
of the i sol ati on of DNA i s gi ven i n one of the practi cal schedul es l ater
(see Chapter 8) and in BASC Booklet 7, Recombinant DNA Technology.
Once isolated, DNA can be broken down or degraded by either: (i)
treatment wi th perchloric acidat 100C, whi ch breaks down the DNA
to i ts consti tuent bases; or (i i ) treatment wi th enzymes(DNase, phos-
phodi esterase, etc.) whi ch break down the DN A to i ts consti tuent
nucleotides.
The mixture of either of these products can be separated by chroma-
tography or el ectrophoresi s and the amount of each of the consti tuent
bases can be measured.
Composition
Usi ng the techni ques outl i ned above i t was noted by Chargaffand co-
workers in the late 1940s that:
(i ) the base composi ti on of an organi sms DNA i s characteri sti c of that
organism;
(i i ) di fferent cel l s or ti ssues of the same organi sm have i denti cal base
composition;
(i i i ) cl osel y rel ated organi sms exhi bi t si mi l ar base composi ti ons (thi s
property is used in biology as a basis for chemical taxonomy)
(iv) the majority of DNA molecules exhibit certain chemical regularities
that are defi ned as Chargaffs base pairingrul es (base equivalence),
namel y the amount of A equal s the amount of T and the amount of G
equal s the amount of C, as can be seen (wi thi n the bounds of experi -
mental error) from the data shown in Table 2.
I n consi deri ng the data shown i n Tabl e 2, i n terms of base equi v-
al ence i t i s necessary to combi ne the val ues for cytosi ne and 5-methyl
cytosine where these apply. The only exception to base equivalence is the
DNA from the virus X174. Can you think of an explanation? (See the
Structure section in this chapter for the solution.)
Structure
I n 1947 Astbury studied DNA using the technique of X-ray diffraction,
shown schematically below.
When the beam of X-rays collides with an electron-dense region in
the DN A, the beam i s di ffracted. From the pattern of the di ffracted
beam i t i s possi bl e to determi ne the arrangement of atoms i n the DNA.
I n this way, Astbury was able to deduce that there was a regular feature
occurring within the DNA molecule every 3.34 . Such a regular feature
is referred to as a crystallographic repeator identity period.
X-ray
source
Diffracted
beams
Photographic
film
DNA
crystal
Table 2.The percentage composition of bases in the DNA from a
variety of organisms
Source of DNA Adenine Guanine Cytosine Thymine 5-Methyl cytosine
Cattle sperm 28.7 22.2 20.7 27.2 1.3
Rat bone marrow 28.6 21.4 20.4 28.4 1.1
Sea urchin 32.8 17.7 17.3 32.1 1.1
Wheat germ 27.3 22.7 16.8 27.1 6.0
Yeast 31.3 18.7 18.1 31.9
Human sperm 30.9 19.9 19.8 29.4
Escherichia coli 26.0 24.9 23.9 25.2
X174 Phage virus 24.3 24.5 18.2 32.3
Later work by Frankl i n, Gosl i n, Wi l ki ns and co-workers (1953)
demonstrated that there were two forms of DN A, a crystal l i ne form
known as the A formand a paracrystalline form known as the B form.
Based on the X-ray di ffracti on work and Chargaffs base equi v-
alence rules, Watson and Crick in 1953 deduced the structure of DNA as
a double helix. Thi s compri sed two strands of sugarphosphate
backbone with the bases positioned in between, with adenine (A) on one
strand bei ng hydrogen (or H-) bondedto a thymi ne (T) on the other,
and a guani ne (G) on one strand bei ng H-bonded to a cytosi ne (C) on
the other. The H -bondi ng arrangement of these base pai rs i s shown
below.
A si ngl e-ri nged pyri mi di ne base opposi te a doubl e-ri nged puri ne
base woul d ensure equal di stance between the two pol y nucl eoti de
strands but why AT and GC speci fi cal l y ? The answer l i es i n the
number of H-bonds that can be formed between the bases as shown (see
also BASC Booklet 7, Recombinant DNA Technology).
N
O
H
H
H
N
N
N N
CH
3
N N
O
N
N
H
H
H
H
N
N N
N
H
N
O
N
O
A T
G C
Sugar
Sugar
Sugar
Sugar
H-bond
Molecular impostor that cannot form hydrogen bonds
In a recent study a synthetic chemical which resembled thymine but which had a
benzene ring instead of the pyrimidine ring and fluorine atoms in place of the
oxygen and nitrogen atoms was used in an assay with DNA polymerase.Much to
the researchers surprise,a DNA helix was produced thus raising doubts on the
role of hydrogen bonds in the DNA structure. A further study showed that mol-
ecular mimics which have the wrong shape distort the helix and that this may rep-
resent the key feature of the structural stability.
New Scientist (22 February 1997)
Because the two pol ynucl eoti de strands have di recti on, two con-
fi gurati ons are possi bl e, one i n whi ch both strands run i n the same
di recti on, whi ch i s known as a parallel arrangement, and one i n whi ch
the strands run i n the opposi te di recti on, whi ch i s known as an
antiparallel arrangement. I t has been shown that the DN A dupl exes
found in nature have their chains in an antiparallel arrangement. This has
a profound si gni fi cance when we come to consi der the bi ol ogi cal
function of the molecules (see DNA replication section).
I n coi l i ng the backbone i nto a hel i cal confi gurati on, two arrange-
ments i n space are possi bl e, one a right-handed helix, the other a l eft-
handed hel i x. The di fference between these two arrangements can be
easily demonstrated using two pipe cleaners. I t has been shown that the
doubl e hel i x of DN A found i n nature i s i n the ri ght-handed confi gu-
ration.
Left-handed Right-handed
Subsequent studi es usi ng space-fi l l i ng model s showed that there
were two groovesi n the DN A mol ecul e, one shal l ow (approx. 12 )
and one deep (approx. 22 ). This feature was found to be important in
rel ati on to the bi ndi ng of certai n anti bi oti cs, pol ymerase enzymes, etc.
The pri nci pal features of the structure of DNA are summari zed i n the
schematic drawing shown below.
Earl i er i n thi s chapter i t was observed that the DNA i sol ated from
the vi rus X174 di d not exhi bi t base equi val ence. The reason for thi s i s
that the DNA of this virus is single-strandedand not the normal duplex
form. This has a significance for its mode of replication.
Triple helices are also possible
Triple helical forms of DNA are now known to exist in vivoand these would
appear to have a key function in certain biological processes,in controlling gene
expression and as tools in genome mapping strategies.Such molecules are now
being actively investigated.
Triple-helical nucleic acids(V.N.Soyfer,1996,ISBN 0-387-94495-8)
DNA as the logic for new breed of computers
The notion of organic computers has been with us for some time now,but
recently the use of short DNA fragments to simulate the logic AND and OR
gates of a computer has been investigated.The claim is that this may now help to
produce a new form of computer that would significantly out-perform those cur-
rently based on silicon.
New Scientist (8 February 1997)
Sugarphosphate
backbone
Hydrogen
bonds
Base
3
3
5
5
Shallow groove Deep groove
1 helical turn 3.4 nm
Breast tissue traps carcinogens
From recent research work it would appear that the fatty tissue that constitutes
80%of womens breasts is capable of absorbing fat-soluble organic molecules,
some of which are carcinogenic,causing DNA damage and mutation.It would
appear that certain foodstuffs may be the source of these carcinogens and as such
it might be impossible or difficult to avoid ingesting these.
New Scientist (7 December 1996)
RNA isolation,base composition and structure
Isolation
The i sol ati on procedure i s very si mi l ar to that for DN A i sol ati on
al though i t i s essenti al to i ncl ude an RNase i nhi bi tor to prevent degra-
dati on of the RNA. Unl i ke DNA, whi ch exi sts as a more or l ess homo-
geneous molecule for any one cell, RNA exists as a family of molecules,
each member of which has a distinctive structure and function (see later
sections in this chapter). Because of this wide variety of RNA species it
is necessary to separate further the initial RNA pool into its constituent
molecules. The methods employed include:
density-gradient centrifugation;
ion-exchange chromatography;
Table 3.DNA structure and function
Structural feature Function
Strong,covalently bonded Preserves the base sequence of genetic
sugarphosphate backbone information during the lifetime of the cell
and allows shortening of the DNA during
chromosome formation in cell division
Complementary purinepyrimidine Keeps an equal distance between the two
base pairing polynucleotide strands,increasing the stability
of the double helix
Numerous but weak H-bonds Enhance stability of the helix but are easily
between complementary bases broken by enzymes to allow DNA replication
and transcription
Large,insoluble molecule Restricts DNA to the nucleus,protecting it
(molecular mass approx.100000 units) from biochemical damage,thus preserving
the genetic code
gel filtration;
electrophoresis.
Base composition
Purified RNA molecules can be degraded by chemical means (alkali) or
by enzymes (RNase) and the base composition determined in a manner
similar to that described for DNA (see section on DNA isolation in this
chapter). One major difference between DNA and RNA is that in RNA
there are a fai rl y l arge number of mi nor bases. Al so, i n the majori ty of
cases there i s no base equi val ence, si gni fyi ng that RNA mol ecul es are
usually single-stranded.
Structure
There are three major forms of RN A: ribosomal RNA (rRN A),
transfer RNA(tRNA) and messenger RNA(mRNA) and these exist in
al l the major l i fe forms, together wi th other di sti nct RN A mol ecul es
whi ch are not uni versal . rRN A accounts for about 80% of the total
cellular RNA and is associated with protein to form the cytoplasmic par-
ti cl es known as ribosomes. As shown bel ow the ri bosome i tsel f can be
consi dered as two subuni ts, a l arge and a smal l , both of whi ch are
RN Aprotei n associ ati ons wi th about 65% RN A: 35% protei n. The
RNA has high molecular mass and is metabolically stable.
Transfer RNA i s the next most abundant speci es and accounts for
about 15% of the total RNA. These mol ecul es are of much l ower mol -
ecul ar mass than the rRNA and are al so referred to as 4S RNA. As we
shal l see l ater, these mol ecul es functi on as adaptorsfor ami no aci ds i n
Front view Side view
30 S subunit
50 S subunit
the course of protei n synthesi s and many di fferent tRNAs exi st, each
being specificfor one amino acid. The tRNA molecule is single-stranded
but the chai n fol ds back on i tsel f i n a very di sti ncti ve way (see di agram
below) such that about 5060% of the structure is base-paired.
Most of the remai nder of the cel l RNA (l ess than 5%) i s accounted
for by mRN A whi ch, i n eukary otes, ori gi nates i n the nucl eus and
mi grates to the cytopl asm duri ng protei n synthesi s (see Chapters 5 and
6). I t is of high molecular mass and is metabolically very labile, i.e. easily
broken down. As we shal l see i n a l ater secti on (see Chapters 5 and 6),
mRNA is centrally involved in the transfer and expression of the genetic
information and is responsible for the sequence of amino acids in each of
the di fferent protei ns i n the cel l . Because the si ze of the messenger i s
variable, no S-value is associated with it.
Note: the S-values refer to how fast a particular molecule sediments in an
ultracentrifuge and the S refers to Svedberg units, the unit of mea-
surement in such studies. Clearly this relates both to the massand shape
of a molecule in that an object of greater mass will move faster than one
with lower mass but the same shape, and one with a small volume will
move faster than one with a similar mass but larger volume (i.e. less
dense).
Loop 3
Loop 1
Loop 2
Anticodon
3
5
RNAs which act as enzymes
In the late 1980s it was shown that certain RNA molecules could act as enzymes,
capable of splicing out specific sequences of RNA either on itself or other RNA
strands.The name given to such molecules was ribozymeand this work earned
a Nobel Prize for one of the researchers (Tom Cech of the University of
Colorado).Two of these ribozymes are now being clinically tested as potential
treatment against HIV (see BASC Booklet 3,Enzymes and their Role in
Biotechnology,for further details).
New Scientist (7 December 1996)
A role for RNA in the treatment of debilitating disease
Specific RNA molecules are being selected from pools of randomly generated and
synthesized RNA as a means of treating the debilitating disease myasthenia gravis.
Sufferers of this disease produce auto-antibodies which block the normal signals
from nerves to muscles by blocking the acetylcholine receptor.It has been shown
that specific RNA molecules are able to bind to the antibodies,so interfering with
their binding to the receptors and hence restoring the normal nerve-to-muscle
signals (see BASC Booklets 5,Immunology,and 9,The Biochemical Basis of Disease
for further information).
Table 4.Differences between DNA and RNA structure and properties
DNA RNA
Deoxyribose sugar Ribose sugar
Thymine base Uracil base
Double-stranded helix Single-stranded molecule
Very large molecular mass Much smaller molecular mass
Insoluble Soluble
3
DNA replication
Learning objectives
explain the biological significance of DNA replication;
explain the suitability of DNA structure for replication;
outline the main features of semi-conservative replication of DNA;
outline the Meselson and Stahl experiment; and
name the major enzymes involved in replication.
Introduction
I n the previ ous chapter i t was shown how we knew that the geneti c
information of a cell is contained in the DNA of that cell. For the cell to
di vi de and produce daughter cel l s i n mi tosi s and mei osi s i t i s essenti al
that the DNA is copied (replicated) and an identical copy is passed to the
daughter cel l . DNA i s repl i cated duri ng i nterphase of both mi tosi s and
meiosis.
The basi c resul ts on whi ch our present day assumpti ons of the
mechanism of DNA replication are built are:
(i ) the evi dence that the geneti c i nformati on was contai ned wi thi n the
nucleic acids;
(i i ) the fact that most DNA consi sts of two strands of pol ynucl eoti de
chains, each of which consists of deoxyribonucleotide residues joined by
35 phosphodiester bonds; and
(i i i ) the fact that H-bonds occur between the bases i n the two strands,
adeni ne i n one strand i s al way s bonded to thy mi ne i n the other and
likewise cytosine with guanine.
21
Each of these we have al ready consi dered, and thus, i f we are gi ven
the sequence of bases i n one strand, we coul d i mmedi atel y wri te down
the sequence of bases in the complementary strand.
Basically, there are five theoretically possible modes of replication.
Conservative
By thi s mechani sm one daughter cel l recei ves the ori gi nal DN A
molecule whilst the other receives a completely new copy.
Semi-conservative
H ere the ori gi nal DN A mol ecul e i s spl i t i nto two strands and each
strand acts as a template on which a new strand is synthesized.
Non-conservative
H ere the ori gi nal DN A i s destroyed compl etel y duri ng the course of
synthesis of two new identical DNA molecules.
Dispersive
H ere the ori gi nal DN A mol ecul e i s di spersed or di stri buted i nto al l
nascent chains.
End-to-end
Here the ori gi nal DNA mol ecul e i s present as hal f of one chai n and the
other half of the complementary chain for both nascent molecules.
Note: A-level students are only expected to consider evidence for or
against the first two possible modes of replication outlined above.
The fi ve possi bl e modes of repl i cati on are shown schemati cal l y i n the
figure which follows.
I n fact it hasbeen proved that DNA replicates semi-conservatively,
fi rst i n E. coli, and subsequentl y i n al l hi gher organi sms as wel l : i t has
been shown to be the case for eukaryotic cells, including human cells, in
22 DNA replication
ti ssue cul ture. The ori gi nal experi ment whi ch proved thi s mechani sm
was carried out by Meselson and Stahl in 1958.
The Meselson and Stahl experiment
(a) Meselson and Stahl grew E. coli in a medium containing
15
NH
4
Cl (i.e.
15
N is the heavy isotope of nitrogen). The result of this was that
15
N was
i ncorporated i nto newl y synthesi zed DNA (as wel l as other ni trogen-
containing compounds).
(b) They al l owed the cel l s to grow for many generati ons i n the
15
N H
4
Cl so that al l the DN A was heavy that i s, had a greater
density than normal. This heavy DNA could be distinguished from the
normal DNA by centri fugati on i n a caesium chloride (CsCl) density
gradient. Note that
15
N is not a radioactive isotope.
(c) They then transferred the cel l s i nto a medi um wi th normal
14
NH
4
Cl and took samples at various definite time intervals as the cells
mul ti pl i ed, and extracted the DNA that remai ned as doubl e-stranded
helices.
Parent DNA
New DNA
Parent DNA
Conservative
Semi-conservative
Non-conservative
End-to-end
Dispersive
The various samples were then run independently on CsCl gradients
to measure the densities of DNA present. The results of this experiment
were as follows:
Thus the DNA that was extracted from the cul ture one generati on
after the transfer from
15
N to
14
N medi um (i .e. after 20 mi nutes) had a
hybrid or intermediate density, exactly half-way between that of DNA
contai ni ng onl y
15
N and that contai ni ng onl y
14
N; each mol ecul e must
therefore have contained one old and one new strand.
DNA extracted from the cul ture after another generati on (i .e. after
40 minutes) was composed of equal amounts of this hybrid DNA and of
l i ght DN A. As ti me went on, more and more l i ght DN A accu-
mulated.
The results of the Meselson and Stahl experiment are not consistent
with non-conservative (only
14
N-DNA bands would be expected from
20 minutes onwards) or conservative (one band of
14
N-DNA and one of
15
N-DNA at 20 minutes would be expected) modes of replication.
Thus far, however, the data are consi stent wi th the three remai ni ng
modes of replication. To distinguish between these, and hence determine
the mode of repl i cati on, Mesel son and Stahl carri ed out an addi ti onal
24 DNA replication
Light
Intermediate
Heavy
0 Generation 1 2 3 4 etc
0
Time interval
following transfer (minutes)
20 40 60 80
D
e
n
s
i
t
y

o
f

m
o
l
e
c
u
l
e
s
a
f
t
e
r

c
e
n
t
r
i
f
u
g
a
t
i
o
n
experi ment i n whi ch they took the DN A i sol ated at 20 mi nutes after
transfer to
14
N -medi um, strand separated thi s and ran the resul tant
sampl e on a CsCl gradi ent. For di spersi ve or end-to-end model s we
would predict a single hybrid band, whereas for semi-conservative repli-
cati on we woul d expect two bands, one l i ght, one heavy. The l atter was
found to be the case and hence DNA replication is a semi-conservative
process.
Mechanism of replication
Watson and Cri ck, i n thei r 1953 paper i n whi ch they proposed the
doubl e-hel i cal structure for DN A, concl ude as fol l ows: I t has not
escaped our noti ce that the speci fi c pai ri ng we have postul ated i mme-
diately suggests a possible copying mechanism for the genetic material .
The i mpl i cati on was that, owi ng to the stri ct compl ementary nature of
the strands, the base sequence of one strand woul d determi ne the base
sequence of the other. I t coul d be i magi ned therefore that repl i cati on
could follow a scheme as shown below.
5
3
3
3
3
3
3
3
3
5
5
5
5
5
5
5
The problem, however, with this scheme is that the energy required
for the first step, namely strand separation, would be far too high to be
plausible. This led to the suggestion that unfolding took place a little at a
time, so forming a replication forkas shown.
Here, as el sewhere i n bi ochemi cal reacti ons, an enzyme i s used as a
catalyst. Enzymes lower the energy needed to activate a reaction, making
i t more l i kel y to occur. For E. coli thi s i s an enzyme cal l ed DNA poly-
merase III. The simplest scheme would be for polymerase molecules to
attach themselves to the ends of the single-stranded DNA template and
to synthesize a complimentary strand of each.
The first problem encountered with this proposal arose because the
polymerase enzyme was found to operate in one direction only, that is it
synthesized the nascent DNA strand in the direction 5!3. This meant
that one of the parent strands (called the leading strand) could be copied
i n a continuousfashi on, whereas the other (cal l ed the lagging strand)
could only be copied in a discontinuousway as a series of short lengths
(approx. 1000 bases l ong) of DNA, each of whi ch i s cal l ed an Okazaki
fragment (see Fi gure bel ow). I t was al so di scovered earl y on that the
DN A pol y merase enzy me coul d not actual l y sy nthesi ze a DN A
molecule from individual DNA nucleotides but instead required a short
stretch of existing DNA helix duplex on which to build. I n this sense it is
truly a DNA-dependentDNA polymerase I I I .
26 DNA replication
3
5
3
3
5
5
RNA
primer
Helix-destabilizing protein
DNA helicase
DNA gyrase
DNA
polymerase III
3
5
The full details of the process of replication are beyond the scope of
the A-l evel syl l abus but i t may be of i nterest to know the rol es of some
of the major enzy mes/protei ns i nvol ved i n the process. The detai l s
required for A-level are given in bold in Table 5.
Exposure to i oni zi ng radi ati on and carci nogeni c chemi cal s, e.g.
phenol s i n ci garette tar, probabl y overwhel m DNA-repai r enzymes
resulting in gene mutation and cancerous cells.
Table 5. Enzymes involved in DNA replication
Enzyme Function
DNA polymerase III The replicase enzyme
DNA polymerase I A repair enzyme involved in removing errors and filling
in gaps in the sequence
DNA ligase Seals nicks in the sugar-phosphate backbone
RNA polymerase Make RNA primers to get the DNA polymerase III started
or RNA primase
RNase H Removes the RNA primers once they have completed
their function
DNA helicase and Unwind the DNA duplex
DNA gyrase
Helix-destabilizing protein Prevents the unwound DNA from immediately rewinding
4
The genetic code
Learning objectives
state that the unit of information is the codon a sequence of three
bases (on DNA and mRNA) representing one amino acid;
state that si nce there are four bases there are 64 possi bl e tri pl et
codons;
state that all codons have been assigned to amino acids or start/stop
signals;
give details of the characteristics of the genetic code; and
predi ct the ami no aci d sequence of a protei n from an mRN A or
DNA base sequence (with the aid of a copy of the genetic code).
Introduction
The mi ddl e to l ate 1950s was a parti cul arl y acti ve and producti ve phase
wi th respect to the research to ai d our understandi ng of the process of
gene expression. At about this time Crick formulated the central dogma
which, in the earliest forms, was expressed as:
Thus far, we have seen that the genetic information which dictates a
cells phenotype is stored in the DNA and this can be passed to progeny
29
Replication DNA RNA Protein
Translation Transcription
cells via the process of replication. We will now consider how this stored
information is expressed to yield the specific phenotype.
A key feature in this respect is the concept of a genewhich has been
defi ned operati onal l y as a regi on of the genomethat segregates as a
si ngl e uni t duri ng mei osi s and gi ves ri se to a defi nabl e phenotypi c trai t
such as a red or whi te eye i n Drosophilaor a round or wri nkl ed seed i n
peas .
The genome is the sum of all DNA nucleotide/base sequences in an
individual organism or species.
Later work demonstrated that a gene or gene regioncorresponds to
a l ength of DNA on the genome that contai ns the i nformati on for the
synthesis of a single protein. This led to the useful concept that one gene
corresponds to one protein.
H owever, as a great many bi ol ogi cal l y acti ve protei ns compri se
more than one pol ypepti de chai n, i t i s more correct to consi der thi s as
one gene one polypeptide chain.
Although serving as a useful memory aid, even this statement is not
enti rel y correct si nce some genes code for si ngl e structural RNA mol -
ecul es, such as mRNA and tRNA, rather than pol ypepti de chai ns and,
also, more recent studies have revealed additional complexities in terms
of the structural organi zati on of the geneti c i nformati on. For the
purposes of the present section, however, it is acceptable to treat the gene
in terms of the one gene one polypeptide chain hypothesis.
Building a human chromosome
The first synthetic chromosome,that of yeast,was built about a decade ago.To
extend this to the synthesis of a human chromosome required detailed consid-
eration of further structural complexities.Thus,in addition to the structural genes
there is a need for telomeres,long repeating DNA sequences which protect the
ends of the chromosome and prevent them binding together,and centromeres,
which provide the scaffolding that enables duplicate chromosomes to split during
cell division.Using this approach,totally artificial chromosomes have been
produced and it is hoped that these may help in gene therapy in the future.
New Scientist (5 April 1997)
30 The genetic code
To transfer the geneti c i nformati on from DN A to a sequence of
ami no aci ds that consti tutes a pol ypepti de chai n i nvol ves two di screte
but consecutive processes:
(i ) transcription, i n whi ch an mRN A strand of nucl eoti des i s sy n-
thesized from one of the strands of the DNA in the gene region; and
(ii) translation, in which the genetic information coded as a sequence of
bases in mRNA is translated or converted into a sequence of amino acids
to form a discrete polypeptide chain.
The gene regions specific to protein synthesis collectively only rep-
resent a small part of the genome for many cells. The remaining regions
are accounted for as non-coding/nonsense sequences, multiple repeat
sequences, control regionsand regions specifying the synthesis of func-
ti onal RNA mol ecul es such as tRNA and rRNA. A summary of these
features is shown below.
How many bases code for an amino acid?
I n the next section we shall consider details of the mechanism by which
the i nformati on coded i n the mRNA i s used to synthesi ze protei ns of a
speci fi c sequence. An i mportant concept underpi nni ng thi s topi c i s the
nature of the code used to translate the sequence of bases in the mRNA
mol ecul e i nto a sequence of ami no aci ds i n a pol ypepti de chai n. Thi s
code is referred to as the genetic codeand in the present section we shall
consider the general properties of the code.
For mRNA to control the synthesis of specific proteins we must ask
how a mol ecul e made up from onl y four di fferent ki nds of base can
determine the order of about 20 different amino acids. Obviously there
cannot be a 1:1 correspondence between the RNA bases and the ami no
Transcription
Control region for gene A Multiple copies of gene C Control region for genes B1 and B2
Translation
gene A
Protein A Protein B (dimer)
Non-coding
sequences
mRNA mRNA mRNA tRNA
gene B1 gene B2
aci ds. Si mi l arl y there cannot be a 2:1 correspondence, si nce thi s onl y
allows for 16 different arrangements (44). I f, however, there are three
bases codi ng for each ami no aci d, the number of possi bl e tri pl ets i s 64
(4 4 4), whi ch woul d be enough combi nati ons to code for each
ami no aci d wi th pl enty to spare. Cl ear-cut evi dence has been produced
that the triplet theoryi s correct and we wi l l now l ook at the evi dence
for such a view. The name given to the sequence of three nucleotides is a
codon.
Evidence for a triplet code
The evidence for a triplet code comes from genetic studies by Crick and
co-workers usi ng mutati ons caused by acri di ne orange or profl avi ne i n
frui t fl i es. The mode of acti on of these compounds i s not ful l y
understood but the net effect i s to cause the i nserti on of an addi ti onal
base i nto a DNA strand or to del ete one of the bases i n a DNA strand,
possibly during DNA replication.
I t i s easi er to expl ai n the resul ts of these studi es by consi deri ng a
simple model and studying its behaviour. I f we assume a DNA sequence
to be a series of triplet codes of bases, or codons, for five amino acids:
CAT CAT CAT CAT CAT
The insertion of a base, T:
CAT TCA TCA TCA TCA
Or deletion of a base, C, from the original sequence:
CAT ATC ATC ATC ATC
The resul t of a si ngl e mutati on (ei ther i nserti on or del eti on) i s to
cause the genetic message/amino acid sequence everywhere to the right
of the mutation to be misread.
The result would almost certainly be a defective protein or enzyme,
especi al l y i f the ami no aci d sequence i s i n or near the acti ve si te of an
enzyme.
Al so i n these studi es i t was shown that i f we i nsert an extra base,
then delete another base a little further on in the sequence, the net result
is an almost identical product (demonstrate thi s for y oursel f). The
important point here is that the insertion and deletion are relatively close
32 The genetic code
together (why ?). There are al l sorts of other way s of combi ni ng
mutation, but the net results provide very strong evidence in favour of a
tri pl et code. These resul ts al so provi de addi ti onal data concerni ng the
mode of translating the code.
Deciphering the code
The probl em of deci pheri ng the code, that i s assi gni ng ami no aci ds to
each of the codons, has been approached usi ng a vari ety of techni ques,
the most productive being the ribosome-binding techniquedevised by
Leder and Ni renberg. Thi s method, l i ke many of the others, makes use
of synthetic mRNA, whi ch i n thi s case i s onl y three bases l ong, i .e. a
si ngl e codon. Cl earl y the advantage of usi ng an mRN A of known
sequence is that it makes data interpretation a relatively straightforward
process.
The ribosome-binding technique involved mixing a sample of pure
syntheti c codon wi th ri bosomes and then, i n turn, each of the di fferent
amino acids (which had been radioactively labelled), each linked to their
correspondi ng tRN A. Each mi xture was fi l tered through a Mi l l i pore
fi l ter whi ch caused the ri bosome and any bound components to be
retained on the filter. The filter could then be tested for radioactivity. I f
we consi der as an exampl e the tri pl et UUG, then i t was found that the
sample which contained radioactive serine did not bind to the filter, and
likewise for the amino acids tryptophan, glycine, methionine, tyrosine,
etc. I n fact, the onl y sampl e whi ch retai ned radi oacti vi ty on the fi l ter
occurred when leucine was used (see diagram below) and therefore it can
be concluded that the codon UUG codes for leucine (Leu).
UUG
UUG
AAC
AAC
Leu* Leu*
Ribosome
Synthetic
mRNA codon
Anti-codon
Amino acid tRNA molecule
Thi s process i s then repeated exactl y as above for a di fferent tri pl et

code. Using this simple yet highly efficient technique (with some minor
modi fi cati ons) i t was possi bl e to assi gn 61 of the 64 possi bl e tri pl ets
unambi guousl y to speci fi c ami no aci ds, and the accepted code i s as
shown here.
Nonsense codons
As has just been seen, 61 of the 64 tri pl ets have been assi gned to par-
ti cul ar ami no aci ds what then i s the functi on of the remai ni ng three,
UAA, UAG and UGA? These were i ni ti al l y l abel l ed as the nonsense
codonsbut it has since been shown that they have an important function
in protein biosynthesis as chain-terminating signals. I t is these codons
whi ch i nform the protei n-synthesi zi ng system that the nascent pol y-
peptide chain has been completely synthesized and to thus allow it to be
released from the ribosome.
34 The genetic code
U
U C A G
C
A
G
U
C
A
G
UUU
UUC
UUA
UUG
UCU
UCC
UCA
UCG
UAU
UAC
UAA
UAG
UGU
UGC
UGA
UGG
CUU
CUC
CUA
CUG
CCU
CCC
CCA
CCG
CAU
CAC
CAA
CAG
CGU
CGC
CGA
CGG
AUU
AUC
AUA
AUG
ACU
ACC
ACA
ACG
AAU
AAC
AAA
AAG
AGU
AGC
AGA
AGG
GUU
GUC
GUA
GUG
GCU
GCC
GCA
GCG
GAU
GAC
GAA
GAG
GGU
GGC
GGA
GGG
U
C
A
G
U
C
A
G
U
C
A
G
} } }
} }
}
}
}
}
}
} }
}
}
}
}
}
}
}
}
}
Phe
Ser
Pro
Thr
Ala Gly Val
Met
Ile
Leu
Leu
His
Gln
Asn
Lys
Ser
Arg
Cys Tyr
OCHRE
AMBER
?
Trp
Arg
Asp
Glu
SECOND LETTER
F
I
R
S
T

L
E
T
T
E
R
T
H
I
R
D

L
E
T
T
E
R
From the experiments just described, plus other related studies, the
general features of the code that are important are as follows:
the code i s sequential (i .e. i t i s read i n stri ct sequence from one end
to the other); and
the code is degenerate.
A most i mportant feature i s that many ami no aci ds are coded by
more than one triplet(all except methionine and tryptophan). I ndeed it
seems that for most ami no aci ds onl y the fi rst two bases of the codon
need be speci fi ed. A code of thi s type i s termed degenerate. I t fol l ows
from thi s that more than one ki nd of tRN A may code for the same
amino acid. This is in fact known to be the case.
The code is universal or ubiquitous.
At present, as a resul t of experi mental data, i t i s general l y accepted
that the code i s uni versal , e.g. Li pmann and others demonstrated usi ng
yeast, Micrococcal or E. coli tRNA, rabbi t reti cul ocyte ri bosomes and
mRN A for haemogl obi n that a perfectl y normal protei n, i ndi sti n-
gui shabl e from wi l d-type, coul d be formed. More recentl y, however,
some excepti ons to thi s general i zati on have been i denti fi ed i n mi to-
chondria, leukaemic cells and in some ciliates, and these observations are
presently posing a challenge for evolutionary theorists.
5
Transcription
Learning objectives
explain the meaning of the term transcription;
explain the role of mRNA;
explain the relationship of mRNA to the DNA template;
outline the necessity of moving the genetic information required to
synthesize proteins from the nucleus to the cytoplasm;
state that the geneti c i nformati on i s carri ed to the cytopl asm by an
mRN A strand whi ch i s compl ementary to the DN A templ ate
strand;
state that onl y a rel ati vel y short segment of DN A codi ng for a
specific polypeptide is transcribed as a unit from the template strand
of DNA;
state that mRNA i s synthesi zed by RNA pol ymerase usi ng ri bo-
nucleoside triphosphates as substrates and DNA as a template; and
outline with the aid of simple labelled diagrams the process of trans-
cription.
Introduction
Transcri pti on i nvol ves the synthesi s of an mRN A mol ecul e compl e-
mentary to a gene regi on/speci fi c nucl eoti de sequence of DN A. Thi s
bei ng the case, we requi re the basi c bui l di ng bl ocks for RNA, namel y
free RN A nucl eoti des and ATP mol ecul es for energy together wi th a
DN A mol ecul e, part of whi ch wi l l be copi ed, the l atter bei ng cal l ed a
DNA template.
37
For such a process to be undertaken at an appropri ate and suffi -
ciently fast rate for the cell requires the help of an enzyme, which in this
case i s cal l ed DN A-dependent RN A pol ymerase or just RNA poly-
merase. Thi s enzy me cannot sy nthesi ze an mRN A strand from free
RNA nucleotides unless a DNA molecule is present.
I t is worth remembering that enzymes have three key advantages.
(i) They act as biological catalyststo speed up biochemical reactions by
lowering the energy needed to activate a reaction. This means a reaction
i s far more l i kel y to proceed under the condi ti ons found i n cel l s, e.g.
moderate temperature and low pressures.
(ii) They usually have high specificityin that one very specific reaction
(or a few rel ated reacti ons) i s catal ysed because of the speci fi c three-
dimensional shape of the active site.
(i i i ) Thei r activity can be controlled, e.g. by thei r products and other
compounds.
Mechanism of transcription
The enzyme RNA pol ymerase, l i ke DNA pol ymerase, operates i n the
di recti on 5!3, that bei ng the di recti on i n whi ch the mRN A i s syn-
thesi zed. Unl i ke DN A pol y merase i t onl y transcri bes one strand of
DNA, i.e. genes are located on a single DNA template strand.
To understand the mechanism of this process it is clearly of interest
to know the way in which the start of each gene is correctly recognized.
The picture which emerged from a study of the DNA sequences near the
start of speci fi c gene regi ons was that of AT-ri ch sequences of bases.
These sequences were si tuated a few bases upstreamfrom the starti ng
point of the mRNA transcript, as shown here.
38 Transcription
Specific gene Base sequence near the start of specific gene region
fd
T7 A2
Lac-UV-5
SV40
E. coli Tyr tRNA
CTGACTATAATAGACAGGGTAAAGACCTG
TGCAGTAAGATACAAATCCGTAGGTAACA
GCTCGTATAATGGTTACAAATAAAGCAAT
CAGCTTATAATGGTTACAAATAAAGCAAT
TTTGATATGATGCGCCCCGCTTCCCGATA
The AT base pair has two H-bonds compared with three for the GC
base pai r (see Chapter 2); and consequentl y a regi on ri ch i n AT pai rs
tends to mel t or strand separate more readi l y, so formi ng a bubble. The
advantages of formi ng thi s bubbl e are that (i ) i t hel ps the RN A pol y-
merase to identify the start of the gene region and (ii) it allows the RNA
pol ymerase to sel ect more easi l y the templ ate or codi ng strand and so
al l ow asymmetri c synthesi s to take pl ace. I n fact i t i s the non-TATA
strand (i .e. the 3!5 strand) that acts as the templ ate strand i n tran-
scription.
Once correct initiationhas been achi eved the RN A pol ymerase
moves al ong the templ ate strand synthesi zi ng an mRN A from RN A
nucl eoti des. The base on each successi ve RN A nucl eoti de i s compl e-
mentary to the DNA template strand.
The mechanisms for terminationof transcription vary from gene to
gene. For some genes an addi ti onal protei n cal l ed rho() i denti fi es the
termi nati on si te and causes the RN A pol y merase to stop sy nthesi s.
Other mechanisms, as shown schematically below, include (i) an AT-rich
regi on fl anked on ei ther si de by a GC-ri ch regi on or (i i ) a palindromic
region. I n both these cases a bubbl e structure i s formed whi ch can be
consi dered to act as a physi cal barri er, so causi ng the RNA pol ymerase
to terminate synthesis as shown.
Each mRN A strand then exi ts the nucl eus through pores i n the
nuclear membrane and attaches to ribosomes in the cell cytoplasm.
CGrich CGrich
AATATTA
TTATAAT
CGATAATCG
GCTATTAGC
A
A
T
AT
T
A
T
T
A
TA
A
T
A
A
T
AT
T
A
T
T
A
TA
A
T
growing mRNA
RNA polymerase
growing mRNA
mRNA
mRNA
T
A
G
C
A
T
C
G
A
G
C
T
A
C
G
A
T
A
T T
T
A
G
C
A
T
C
G
G
C
T
A
C
G
A
T
(i)
(ii)
The mechanism of transcription is summarized schematically below.
40 Transcription
T
A
T
A
T
A
T
A
T
A
T
A
T
A
T
A
T
A
T
A
Sense strand
RNA polymerase
Gene
Initiation site
DNA unzipped by
RNA polymerase
Termination sequence
Growing mRNA transcript
mRNA
5
6
Translation
Learning objectives
state that ribosomes are responsible for the translation of the genetic
i nformati on encoded as a sequence of bases i n the mRN A strand
into a specific sequence of amino acids in a polypeptide chain;
outline the functions of ribosomes, mRNA, tRNA and amino acids
in translation; and
outline the processes involved in translation using the terms: codon,
anti codon, pepti de-bond formati on and termi nati on, usi ng si mpl e
diagrams.
Introduction
I t can be shown that no specific affinity/binding occurs between amino
acids and the mRNA, to neither the sugarphosphate backbone nor the
bases (see the Figure below). This being the case, we cannot have a direct
read-off in translation as we did in replication and transcription.
The sol uti on to thi s probl em i s to have an i ndi rect associ ati on
achi eved by coupl i ng the speci fi c ami no aci d to the mRN A vi a an
adaptor molecule, which turned out to be tRNA.
41
mRNA
Amino acid
No reaction
Binds to mRNA
Binds to amino acid
mRNA
tRNA
The tRNA mol ecul es must be acti vated before they can attach to a
speci fi c ami no aci d and perform thei r rol e i n transl ati on of the geneti c
code.
Activation of tRNA
To bind the amino acid to the tRNA molecule requires ATP energy and
a specific enzyme called an amino acyl-tRNA synthetase. The reaction
occurs i n two stages: i n the fi rst the ami no aci d i s acti vated and i n the
second the acti vated ami no aci d i s transferred to i ts speci fi c tRN A as
shown schematically below.
42 Translation
A
A
Amino acid
Enzyme (amino acyl-
tRNA synthetase)
ATP
Pyrophosphate
Phosphate
A
A
C
C
A
tRNA
C
C
A
AMP
C
C
A
Amino acyl-tRNA
(an activated
amino acid)
The amino acyl-tRNA synthetase enzyme, apart from catalysing the
reacti on, al so di spl ays hi gh speci fi ci ty i n that i t wi l l onl y bi nd a par-
ti cul ar ami no aci d to i ts correct, speci fi c tRNA mol ecul e thus ensuri ng
hi gh accuracy /fi del i ty i n the transl ati on event. The ami no aci ds are
l i nked to the 3 end of the tRNA (al l end wi th the sequence CCA-OH)
via their carboxyl group (COOH).
Initiation
Translation of the mRNA strand starts from the 5 endat a specific ini-
tiation siteor triplet codon, AUG, which normally codes for the amino
aci d methi oni ne. Thi s i ni ti ati on si te AUG codon i s di sti ngui shed from
all other AUG sequences as it is preceded in the mRNA sequence by an
AGGA base sequence, known as the ShineDalgarno consensus
sequence, named after the discoverers.
Thi s sequence i s recogni zed by a UCCU compl ementary sequence
in the tRNA of the small ribosomal subunit and so causes the ribosome
to bind to the correct starting point on the mRNA as seen below.
I t was al so shown that thi s uni que AUG bi nds a speci al ami no aci d
(linked to its corresponding tRNA as described previously). This special
ami no aci d i s a methi oni ne resi due i n whi ch the ami no group has a
formyl group (CHO) bound to i t and i s cal l ed N-formylmethionine
(fMet) i n prokaryotes. The advantage of thi s added compl exi ty i s that
thi s effecti vel y bl ocks the ami no group and i nhi bi ts i t reacti ng, thereby
directing the polypeptide chain to be synthesized in the N-terminal!C-
terminal direction. Eukaryotes do not use the formyl group.
AGGA
AUG AUG
AUG AUG
AGGA
UCCU
AUG AUG
AUG AUG
5
5
3
3
By such means an initiation complex i s formed i n whi ch the
ribosome is bound to the correct site on the mRNA and the first amino
acid (linked to its corresponding tRNA) is in position.
Elongation
After thi s the next ami no aci d speci fi ed by the codon fol l owi ng the
AUG sequence (agai n an ami no acy l -tRN A) i s bought i nto posi ti on
alongside the first residue in order that these can be linked together via a
peptide bond, an exampl e of a strong coval ent bond. The energy
requi red for the formati on of a pepti de bond comes from the hi gh-
energy bond between the tRN A mol ecul e and i ts speci fi c ami no aci d
produced by the breakdown of ATP molecules.
The condensation of (or peptide bond between) the two amino acids
i s catal ysed by an enzyme cal l ed peptidyl transferase, one of the con-
sti tuent protei ns of the RNAprotei n compl ex that makes up the l arge
ri bosomal subuni t. I n thi s way, the fMet i s transferred on to the other
ami no acy l -tRN A and i ts tRN A (cal l ed a spent tRNA) l eaves the
compl ex. The ri bosome moves al ong three bases by a process cal l ed
translocationand thi s al l ows the next ami no acyl -tRNA to bi nd to i ts
codon on the mRNA. The individual amino acyl-tRNA molecules bind
to the correct codon sequences by vi rtue of each tRNA havi ng a three
base sequence, known as the anticodon, which is complementary to the
codon sequence and so H -bonds to i t. Because onl y a speci fi c tRN A
mol ecul e attaches to the mRNA codon the correct ami no aci d i s pl aced
i n the growi ng pol ypepti de sequence. The growi ng chai n i s transferred
to the i ncomi ng ami no acyl -tRN A vi a a second pepti de bond and, as
44 Translation
fMet
OH
Spent tRNA
tRNA
molecules
Amino acids
Peptide
bond
aa
2
aa
2
fMet
before, the spent tRN A l eaves the compl ex because of the rel ati ve
weakness of the H-bonds between the codon and anticodon bases.
Termination
The above process conti nues exactl y as descri bed unti l the ri bosome
encounters one of the codons referred to earlier as the nonsense codons
(see Chapter 4). As those codons do not code for speci fi c ami no aci ds
thi s serves as the si gnal to termi nate the process and consequentl y the
ri bosome di ssoci ates from the mRN A and the pol y pepti de chai n i s
hydrolysed from the tRNA to which it is currently bound.
Once protei n synthesi s i s under way, the formyl group i s cl eaved
from the methi oni ne by an enzyme cal l ed deformylaseand, for many
proteins, a number of amino acids are also removed from the N-terminal
end by peptidaseenzymes. This type of processing is referred to as post-
translational modification. The pol ypepti de chai n wi l l now fol d up
i nto the secondary and terti ary structure of the functi onal protei n/
enzyme and is transported to its required location in the cell through the
rough endoplasmic reticulum membrane system.
Ribosome
Start
mRNA
3 end
5 end
Complete polypeptide
released
Ribosome
subunits released
Stop
Growing polypeptide
Therefore, by the processes of transcri pti on and transl ati on the
l arge, permanent, i nsol ubl e DNA mol ecul e, whi ch i s restri cted to the
nucl eus, can control the expressi on of i ts geneti c code by produci ng a
protei n/enzyme mol ecul e whi ch can now affect a bi ochemi cal process
throughout the cell.
Polysomes
I n the living cell, each strand of mRNA usually carries several ribosomes
at any one ti me, each at a di fferent stage i n the formati on of the protei n
whi ch i s bei ng synthesi zed. Such groups of ri bosomes strung out on a
thread of mRNA are known as polyribosomesor polysomes, as shown
here in the electron micrograph.
The pol ysome mechani sm protects the mRNA mol ecul e, del ayi ng
i ts breakdown, and greatl y i ncreases the effi ci ency of transl ati on, i .e.
many protei n mol ecul es can be produced from a si ngl e mRNA strand,
thus saving the cell energy.
Pol ysomes are found both free i n the cytopl asm and cl osel y asso-
ciated with the outer face of the rough endoplasmic reticulum membrane
system.
Recreating an extinct bird
Groups of researchers in Japan and New Zealand are studying the genetic
makeup of the giant moa,a flightless bird which became extinct about 300 years
ago.These workers have isolated DNA from the femur of a giant moa and plan to
introduce these into chicken embryos.The area of particular interest is the
homeobox,which controls embryonic development by switching specific genes
on or off.It is hoped in this way to switch on common ancestor genes and to see
if they can reproduce some of the features of the extinct moa.
46 Translation
260000
7
Assessment:
past examination questions
and outline solutions
As stated earl i er, i t i s hoped that the i ncl usi on of detai l ed l earni ng
objectives within each section of these guidance notes will not only help
students to i denti fy key topi cs i n the sy l l abus but al so hel p them to
gauge the level of expected outcome, and hence be of value in the formal
assessment of the material.
Many of the Exami nati on Boards publ i sh detai l ed anal yses of the
exam-questi on performance and i t may be worthwhi l e to read these
carefully and modify the guidance notes where appropriate to reflect the
varying emphasis placed on the different themes within this broad topic.
The fol l owi ng questi ons are actual A-l evel Bi ol ogy exami nati on
questi ons together wi th thei r sol uti ons provi ded by the authors. Other
sol uti ons and approaches may al so be val i d. Presented i n thi s way they
might serve as useful source material in the review of each of the sections
at the end of formal l essons. Once agai n, we are grateful to the vari ous
Exami nati on Boards, especi al l y the N orthern Exami nati ons and
Assessment Board, for al l owi ng past exami nati on questi ons to be used.
Answer all the questions in the spaces provided.
Question 1
The diagram shows the sequence of bases on one strand of a short length
of DNA. This sequence should be read from left to right.
CGACCCCAG
47
(a) Give:
(i ) the base sequence that wi l l be produced as a resul t of trans-
cription of the complete length of DNA shown in the diagram;
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
(i i ) the three bases of the tRN A that wi l l correspond to the
sequence of bases shown by the underlined bases on the diagram.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1)
As a resul t of a mutati on, the fi rst base i n the l ength of DNA shown i n
the diagram is lost (deleted).
(b) (i ) Use Tabl e 6 to i denti fy the fi rst two ami no aci ds for whi ch
the mutated DNA codes;
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1)
(i i ) expl ai n why mutati ons i nvol vi ng the del eti on of a base may
have greater effects than those i nvol vi ng substi tuti on of one
base for another.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
Question 2
I n an i nvesti gati on of DN A repl i cati on, a speci es of bacteri um was
grown for many generations in a medium containing the
15
N isotope of
nitrogen, rather than its more common form,
14
N.
DNA was isolated from the bacterial cells and separated into single
strands by mild chemical treatment. The density of this single-stranded
DN A was measured usi ng densi ty -gradi ent centri fugati on. I n thi s
48 Past examination questions
Table 6.DNA base sequences and the amino acids for which they
code
DNA base sequence Amino acid
ACC Tryptophan
CAG Valine
CCA Glycine
CCC Glycine
CGA Alanine
GAC Leucine
techni que, sampl es of di fferent densi ti es settl e at di fferent l evel s i n the
tube. Some of the results obtained by using this technique are shown in
the Figure. All samples were treated in the same manner.
(a) Densi ty-gradi ent centri fugati on enabl es di fferent types of DNA
to be di sti ngui shed. Expl ai n how the use of the
15
N i sotope i n
this experiment makes this possible.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
(b) Account for the resul ts obtai ned i n Tube C, expl ai ni ng why no
DNA of intermediate density was observed at this stage.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3)
(c) Bacteri a l i ke those that gave the resul t shown i n Tube C of the
Fi gure were grown for a further generati on on a medi um con-
tai ni ng
14
N . DN A was i sol ated from these bacteri a and treated
as in the first investigation.
Show, on the fol l owi ng Fi gure, the resul t expected wi th densi ty-
gradi ent centri fugati on. Al so, i ndi cate the rel ati ve amount of
each type of DNA you would expect to be present.
Tube C Tube B
Tube A
DNA
X
DNA from
bacteria
grown on
14
N
DNA from
bacteria
grown on
15
N
DNA from bacteria
grown originally on
15
N,
but then transferred for
one generation to a medium
containing
14
N
Sugar
solution
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
Studies were carried out to determine the nitrogenous base composition
of DN A i n thi s bacteri um. Thi s i nvol ved fi ndi ng val ues for doubl e-
stranded DNA as well as for each individual (and ) DNA strand.
The Table below gives some of the results obtained.
(d) Enter the seven missing values in the table. (4)
(e) What rati o of (AG) to (CT) woul d you expect i n the band of
DN A marked as X i n Tube C i n the fi rst Fi gure? Expl ai n your
answer.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
Question 3
Al bi no mi ce are compl etel y whi te because they cannot produce coat
pigment. They cannot produce this pigment because of a gene mutation
that affects the formati on of an enzyme i n the metabol i c pathway that
DNA from bacteria
grown originally on
15
N,
but then transferred for
two generations to a
medium containing
14
N
Table 7. Base composition of DNA
Percentage of base
present in DNA sample Ratio of (AG) Ratio of (AT)
DNA sample A G C T to (CT) to (CG)
Double-stranded 30 20 20 1.00 1.5
Strand 28 26 32 1.17
Strand 32 0.85
produces the pi gment. The enzyme produced by the mutant gene does
not function effectively.
(a) (i) Explain what is meant by gene mutation;
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1)
(i i ) expl ai n how a gene mutati on coul d resul t i n a change i n the
ami no aci d sequence i n the enzy me produced by the coat-
pigment gene.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4)
(b) Expl ai n why mi ce that are heterozy gous for thi s mutant gene
are never albinos.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
Question 4
The diagram shows part of a DNA molecule.
(a) How are the two strands of the DNA molecule held together?
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1)
(b) What do each of the following letters on the diagram represent?
W. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Z. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4)
Y
guanine
W
Z
X
thymine
Question 5
One techni que used to produce human i nsul i n by geneti c engi neeri ng
involves inserting a gene for human insulin into the DNA of a bacterium.
(a) Name the enzyme that would be used to:
(i) cut the bacterial DNA;
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1)
(ii) insert the DNA for human insulin into the cut bacterial DNA.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1)
(b) There are 51 ami no aci ds i n i nsul i n, made up of 16 ami no aci ds
out of the 20 that are coded for by DN A. What i s the mi ni mum
number of di fferent ty pes of tRN A mol ecul e necessary for the
synthesis of insulin? Explain your answer.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
(c) The base sequence bel ow i s part of the DN A sequence that
codes for insulin.
CCATAGCAC
(i) Write down the corresponding mRNA sequence;
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1)
(i i ) A mutati on occurred that repl aced guani ne i n thi s DN A
sequence wi th a di fferent base. Expl ai n the possi bl e effects of
this mutation on the structure of the insulin molecule.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3)
Question 6
Scientists have shown that kidney beans are resistant to cowpea weevils
and adzuki bean weevi l s, two of the most seri ous pests of Afri can and
Asi an pul ses (vegetabl es rel ated to peas and beans). Thi s i s because the
beans produce a protei n that i nhi bi ts one of the weevi l s di gesti ve
enzy mes. Weevi l s that eat the pul ses soon starve to death. The
researchers have i denti fi ed the gene that produces the i nhi bi tor and
removed i t from the ki dney bean DN A. They i nserted the gene that
produces the i nhi bi tor i nto the DN A of a bacteri um cal l ed Agro-
bacterium tumefaciens. Using this bacterium they have been able to add
the inhibitor gene to peas. They hope soon to be able to add the gene to
African and Asian pulses.
(a) Describe how scientists could:
(i) remove the gene that produces the inhibitor from kidney beans;
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
(ii) insert this gene into the DNA of a bacterium.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
(b) The DN A i n the bacteri um i s abl e to repl i cate to produce many
copi es of i tsel f for i nserti on i nto pea cel l s. Descri be the structure
of a DN A mol ecul e and expl ai n how thi s structure enabl es the
molecule to replicate itself.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (8)
Question 7
(a) Draw a l abel l ed di agram of a representati ve l ength of the DN A
(deoxyri bonucl ei c aci d) mol ecul e (the chemi cal structure of the
different constituents is not required). (3)
(b) N ame the repeati ng uni ts that are joi ned to form the macro-
molecule of DNA.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1)
(c) Suppose that a secti on of DN A has the fol l owi ng sequence of
bases:
GGATTAACACCT
What wi l l be the compl ementary messenger RN A (ri bonucl ei c
acid) sequence?
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
(d) Usi ng the Tabl e of ami no aci d tri pl ets bel ow, determi ne the
sequence of the ami no aci ds of the protei n synthesi zed from thi s
messenger RNA.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
(e) N ame the twoother ki nds of RN A that parti ci pate i n protei n
synthesis.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
Question 8
I n 1958, Mesel son and Stahl publ i shed the resul ts of an experi ment
whi ch provi ded strong evi dence that cel l s produce new DN A by a
process of semi-conservative replication.
(a) Why is replication of DNA described as semi-conservative?
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
Table 8.Selected amino acids and their related mRNA triplet
codes
Triplet code Amino acid
AAU Asparagine
CCU Proline
GAA Glutamic acid
AUG Methionine
CUA Leucine
GGA Glycine
ACA Threonine
GCU Alanine
UGU Cysteine
Mesel son and Stahl s experi ment i s outl i ned i n the fol l owi ng
Figure (
15
N is a heavy isotope of nitrogen).
(b) Which component of the DNA was labelled with the
15
N?
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1)
(c) Expl ai n why centri fugati on separates the DN A l abel l ed wi th
different isotopes of nitrogen.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
(d) On the di agram above, draw i n the resul ts you woul d expect for
generations 2 and 3.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
Question 9
Read the following passage.
For more than 30 years, Professor Stanley Miller of the University of Calif-
ornia has been trying to show how life began. From a primitive mixture
of hydrogen, ammonia, methane and other simple chemicals exposed to
lightning and ultraviolet radiation, he has been trying to produce a self-
replicating chemical that might have been the first form of life.
His latest experiments support the view that early life was based on
RNA, rather than DNA to which it is closely related. RNA still plays a
central role in life as the agent for transferring the information stored in
DNA is extracted and centrifuged
E. coli with
15
N-labelled
DNA grown
on
14
N medium
Cells
replicate
once on

14
N medium
Cells
replicate
a second
time on

14
N medium
Cells
replicate
a third
time on

14
N medium
0 Generation 1 2 3
DNA and used to make proteins. I n early life it seems to have been more
i mportant sti l l . RNA can not onl y act as a store of geneti c i nformati on
and a means of transferring it, but can also act as a catalyst.
Professor Mi l l ers l atest experi ments go back to an earl i er stage of
the process, seeki ng to show how the RN A i tsel f mi ght have been
produced. H e has shown that i t i s l i kel y that al l the components on
whi ch RN A i s based coul d have been made i n the condi ti ons on the
Earth 3.5 billion years ago.
Adapted from an article by Nigel Hawkes in The Times(3 July 1995).
(a) I n what way s are the structures of RN A and DN A al i ke, and i n
what ways are they different?
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4)
(b) Expl ai n how RNA acts as the agent for transferri ng the i nform-
ation stored in DNA that is used to make proteins (lines 7 and 8).
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (6)
(c) Suggest why a nucl ei c aci d mol ecul e woul d have been essenti al
in the first forms of life.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)
Answers
Question 1
(a) (i) GCUGGGUC.(2 marks if fully correct; 1 mark if only G and C correct)
(ii) CAG. (1)
(b) (i) Leucine and glycine. (1)
(i i ) Del eti on affects al l (subsequent) tri pl ets i n the DNA sequence and so
may al ter enti re protei n; substi tuti on may al ter onl y a si ngl e ami no
aci d/tri pl et codon; i f the thi rd base i n the tri pl et changed, i t may have no
effect/degenerate code. (any 2)
Question 2
(a)
15
N is incorporated into (nitrogenous) base of DNA. I ts presence increases
the density of the DNA. (2)
(b) The DNA i n Tube C consi sts of a dense
15
N strand, the templ ate used i n
repl i cati on. Al so present i s a l ess-dense
14
N strand, the newl y repl i cated
56 Outline solutions
strand. No intermediate exists because the DNA was made single stranded
before it was centrifuged. (3)
(c) There will be two bands present, one of lower density (top of the tube) and
one of hi gher densi ty. The former contai ns about three ti mes as much
DNA as the latter. (2)
(e) 1.00, because it is composed of equal numbers of and strands. (2)
Question 3
(a) (i) Change in sequence of bases/nucleotides in gene/codon. (1)
(i i ) Any four of the fol l owi ng: (substi tuti on) l eadi ng to di fferent codon/
bases at this point (in DNA) chain; leads to wrong amino acid insertion at
thi s poi nt i n pol ypepti de; del eti on/addi ti on al ters al l subsequent ami no
aci ds; l eads to changed sequence of al l subsequent ami no aci ds; copy of
changed (codon) i n mRN A; reference to ri bosomes; reference to anti -
codons on tRNA; reference to specific amino acid attached to tRNA. (4)
(b) Heterozygotes always have one dominant allele, therefore one DNA chain
will always produce enough functional enzyme. (2)
Question 4
(a) Hydrogen bonding/specific base pairing. (1)
(b) Wphosphate or phosphoric acid group; Xdeoxyribose/sugar/pentose;
Ycytosine; Zadenine. (4)
Question 5
(a) (i) Restriction enzyme/endonuclease/named enzyme e.g. EcoR1, HindI I I ,
BamHI . (1)
(i i ) DNA l i gase (see BASC Bookl et 7, Recombinant DNA Technologyfor
further details). (1)
(b) 16, because each tRNA is specific to one amino acid. (2)
(c) (i) GGUAUCGUG; (1)
(ii) any four of the following: might code for a stop codon; might code for a
di fferent ami no aci d; thi s woul d make a modi fi ed protei n; protei n mi ght
not functi on; ami no aci d mi ght not al ter (because the change occurs
on third triplet); therefore no change to protein made. (3)
Question 6
(a) (i) Restriction enzyme/endonuclease. Removes section of DNA. (2)
(i i ) Two of the fol l owi ng: cut bacteri al DN A/pl asmi d; l i gase; spl i ces i n
DNA; reference to vectors, e.g. phage. (2)
(See BASC Booklet 7, Recombinant DNA Technologyfor further details.)
(b) Ei ght of the fol l owi ng (al l ow poi nts on di agram i f appropri atel y
labelled/annotated): reference to nucleotides; four bases named; reference
to sugarphosphate backbone; reference to two (polynucleotide) strands;
(d) Percentage of base
present in DNA sample Ratio of (AG) Ratio of (AT)
DNA Sample A G C T to (CT) to (CG)
Double-stranded 30 20 20 30 1.00 1.5
Strand 28 26 14 32 1.17 1.5
Strand 32 14 26 28 0.85 1.5 (4)
reference to speci fi c base-pai ri ng; exampl e, e.g. AT/CG; reference to
bonding/hydrogen bonds; reference to uncoiling/unzipping; reference
to new complementary strand formation. (8)
Question 7
(a) Phosphate/sugar/base: in correct sequence; antiparallel strands. (3)
(b) Nucleotides. (1)
(c) CCUAAUUGUGGA (2; 1 mark if single error)
(d) Proline, asparagine, cysteine and glycine (2; 1 mark if single error)
(e) Transfer RNA and ribosomal RNA. (2)
Question 8
(a) Both daughter hel i ces contai n one (conserved) strand from the parent
molecule and one newly synthesized complementary DNA strand. (2)
(b) The nitrogenous (nitrogen-containing) bases. (1)
(c) The
15
N DNA has a hi gher densi ty than that of
14
N and the di fference i n
buoyant densi ty i s suffi ci entl y great to be abl e to be resol ved as separate
bands in a caesium chloride gradient,
15
N moves further down in gradient. (2)
(d) Generati on 2 woul d have two bands of equal i ntensi ty, one at the hybri d
densi ty and one at the l i ght densi ty. Generati on 3 woul d al so have two
bands at the same densities but the ratio of amount of DNA would be 3:1,
light:hybrid. (2)
Question 9
(a) Alike: contain the same bases adenine, guanine and cytosine; basic building
bl ocks of nucl eoti des consi st of sugar, phosphate and base; nucl eoti des
linked by phosphodiester bonds.
Different: DNA contains the base thymine whilst RNA contains the base
uraci l ; DN A contai ns the sugar deoxyri bose whi l st RN A has the sugar
ri bose; DNA i s usual l y doubl e stranded and l arge; RNA i s usual l y si ngl e
stranded and relatively small. (4; maximum of 2 for each section)
(b) Specific portion of the DNA helix coding for the protein unzips; comple-
mentary RNA nucleotides line up opposite exposed DNA bases; template
DN A strand i s used; mRN A strand sy nthesi zed by DN A-dependent
RNA polymerase; mRNA strand released and exits nucleus through pore
i n membrane; attaches to ri bosomes (contai ns rRN A); each tRN A
mol ecul e carri es a speci fi c ami no aci d; ri bosomes move al ong mRN A
strand; codon/anti codon i nteracti on ensures correct sequence of ami no
aci ds; adjacent ami no aci ds joi ned by pepti de bond to produce
polypeptide/protein. (any 6)
(c) The need to have a stabl e, compact store of i nformati on, a sel f-repl i cati ng
molecule which can mutate to allow evolutionary survival of the fittest. (any 2)
58 Outline solutions
8
Laboratory practicals
Extraction and characterization of DNA from
Micrococcus lysodeikticus
As DNA i s a l arge fragi l e mol ecul e, i sol ati on methods must be mi l d i n
order to avoi d excessi ve breakage of DNA chai ns. Extreme condi ti ons
(ei ther of pH or temperature) must be avoi ded and the fol l owi ng pro-
cedure describes a method of DNA preparation from a bacterial source.
Students should record carefully any changes which occur at each stage
of the method and try to account for any changes.
Method (day 1,2hours)
Suspend 0.5 g of freeze-dried Micrococcus lysodeikticuscells in 20 ml of
0.15 M N aCl /0.1 M EDTA, pH 8.0. Add 20 mg of l y sozy me and
i ncubate at 37C for 15 mi n. Add 1.6 ml of 25% (w/v) sodi um dodecyl
sulphate (SDS) solution, mix and incubate for 10 min at 65C. Cool and
add 5.5 ml of 5 M sodium perchlorate followed by 25 ml of chloroform-
i soamyl al cohol (25:1 v/v). Mi x thoroughl y for 15 mi n. Centri fuge for
10 mi n at hi gh speed. Careful l y draw off the top aqueous l ayer wi th a
Pasteur pipette and place the solution in a narrow beaker. Carefully layer
on top an equal volume of ice-cold absolute alcohol. Using a clean glass
rod collect the DNA precipitate from the interface and transfer to a clean
porti on of ethanol . Wash the DNA by unwi ndi ng and rewi ndi ng onto
the rod. Transfer to a tube containing 3 ml of SSC (0.15 M NaCl/0.015M
sodium citrate, pH 7.0). Leave until next practical period to dissolve.
59
Properties of DNA
Hyperchromicity
Thi s feature of DN A ari ses from the fact that when doubl e-stranded
DNA is denatured (i.e. strands are separated) the absorbance at 260 nm
increases substantially (up to 40%).
Base composition
The base compositions of DNA and RNA differ, and this can be demon-
strated by chromatography of hy drol y sates of sampl es of each. By
el uti ng the separated bases and determi ni ng the amount of each spec-
trophotometrically the base ratios can be calculated and compared.
Method (day 2,2.5hours)
Make a 1-i n-10 di l uti on of 1 ml of your DN A sampl e usi ng SSC. I f a
spectrophotometer i s avai l abl e record the ul travi ol et absorpti on
spectrum using SSC as blank (further dilution may be necessary). Heat a
sampl e of your di l ute DNA to 100C for 15 mi n and cool qui ckl y. Re-
record the spectrum.
Pl ace approx. 20 mg of DNA and RNA provi ded and your DNA
sol uti on [an A
260
of 1 opti cal densi ty (OD) uni t i s approx. 50 g/ml ] i n
separate cl ean test tubes and add 0.5 ml of 70% H Cl O
4
. H eat i n a
boi l i ng-water bath for 1 h i n a fume cupboard. Cool , add an equal
volume of water, and spot 10 l samples on to a chromatogram together
wi th 10 l sampl es of thymi ne, adeni ne, guani ne, cytosi ne and uraci l
solutions. Develop the chromatogram using 65% isopropanol/16% con-
centrated HCl/19% water as solvent.
Method (day 3,1.5hours)
Exami ne the chromatogram under ul travi ol et l i ght and mark i n penci l
l i nes around the spots (wear a face mask!). Measure R
F
val ues and cut
out the separated bases wi th sci ssors (i ncl udi ng approx. equal areas of
the chromatograms as a blank). Transfer to clean test tubes and elute the
bases by addi ng 10 ml of 0.1 M H Cl . H eat to 45C for 1015 mi n and
allow to cool. Record the ultraviolet absorption spectrum of each eluate
and calculate concentrations using molar extinction coefficients.
60 Laboratory practicals
Purification of RNA
Before use i n bi ochemi stry practi cal cl asses, commerci al l y avai l abl e
RNA must first be purified and treated in the following sequence:
(i) 10 g of RNA (BDH-yeast) taken in some distilled water and dissolved
by bringing to pH 6.87.0 with NaOH;
(ii) dilute to 200 ml with distilled water;
(i i i ) spi n down i nsol ubl e resi due i n a centri fuge (5000 revol uti ons/mi n
for 15 min);
(iv) dialyse for 48 h in three changes of 2 litres of ice-cold distilled water;
(v) preci pi tate wi th 2 vol s. of 95% ethanol and sol i d sodi um acetate to
make 2% solution;
(vi) spin down RNA, redissolved to 200 ml with distilled water;
(vii) dialyse for 24 h against distilled water;
(viii) precipitate with 2 vols. of 95% ethanol and bring to 2% with solid
sodium acetate;
(ix) RNA spun down and wash the precipitate twice with ethanol:ether
(1:1);
(x) wash precipitated RNA with ether and air dry (stirring well).
Store cold and use within 8 weeks.
Further experiments relating to the physical and chemical properties
of DNA and RNA can be performed using the DNA isolated in this way
or usi ng materi al obtai ned from commerci al sources. The choi ce of
experiments is best made by the individual teacher based on such factors
as avai l abi l i ty of speci al i st equi pment, cost of consumabl es, safety and
practi cal ti me avai l abl e. Ti tl es for some practi cal s i n thi s area are gi ven
bel ow together wi th the appropri ate reference. Practi cal s rel ati ng to
functi onal aspects of the nucl ei c aci ds that are sui tabl e for school are
sparse, l argel y because many depend on the use of very speci al i st
equi pment or expensi ve and potenti al l y hazardous radi ochemi cal s. I n
these cases use may be made of computer simulations and/or videotape
materi al and possi bl e ti tl es are i ncl uded bel ow and i n the l i st that
fol l ows. School teachers may al so wi sh to take the opportuni ty of
l i nki ng wi th a nei ghbouri ng i nsti tute of hi gher educati on i n order to
establish access to specialist equipment of this type. At the present time
many such institutions would welcome such an approach.
Further practicals
Falconer,A.C.and Hayes,L.J.(1986) The Extraction and Partial Purification of Bacterial DNA as
a Practical Exercise for GCE Advanced Level Students.Journal of Biological Education20,
2526
Wood,P.(1984) Use of Computer Simulations in Microbial and Molecular Genetics.Journal of
Biological Education20,309312
Goodridge,F.(1983) The Teaching of Protein Synthesis a Microcomputer Based Method.
Journal of Biological Education17,222224
New,J.and Jolley,R.(1986) Ultraviolet Radiation Induction of Mutation in Penicilliumclaviforme.
Journal of Biological Education20,155157
Franco,R.and Canela,E.I.(1985) SIMCODE;A Program for Simulating Point Mutations in
Genomic DNA.Biochemical Education13,6667
Spinder,S.,Siebert,P.,Coffman,F.and Jurnak,F.(1984) Isolation of Biologically Active mRNA.
Biochemical Education12,2225
Rosenfield,I.Z.H.E.and Van Loon,B.(1983) DNA for Beginners,Writers and Readers Publishing
Co.,location ISBN 0 86316 023 9
62 Laboratory practicals
9
Source materials
Textbooks
Boyer,R.F.(1986) Modern Experimental Biochemistry,AddisonWesley Publishing Co.Inc.,
Wokingham [experiments 1520]
Calladine,C.R.and Drew,H.R. (1997) Understanding DNA(2nd edition),Academic Press,
London
Campbell,N.A.(1996) Biology(4th edition),Benjamin/Cummings Publishing Company,California
Drlica,K.(1984) Understanding DNA and Gene Cloning:A Guide for the Curious,John Wiley and
Sons,Chichester
Garrett,R.H.and Grisham,C.M.(1997) Biochemistry,Saunders College Publishing,California
Matthews,C.K.and van Holde,K.E.(1996) Biochemistry (2nd edition),Benjamin/Cummings
Publishing Company,California
Matthews,H.R.,Freedland,R.and Miesfeld,R.L.(1997) Biochemistry: A Short Course,John Wiley
and Sons,Chichester
Rosenfield,I.,Riff,E.and Van Loon,B.(1983) DNA for Beginners,Writers and Readers Publishing
Co., ISBN 0 86316 023 9
Sinden,R.R.(1994) DNA Structure and Function,Academic Press,London
Solomon,E.P.,Berg,L.R.,Martin,D.W.and Villee,C.(1996) Biology(4th edition),Saunders College
Publishing,California
Voet,D.and Voet,J.G.(1995) Biochemistry (2nd edition),John Wiley and Sons,Chichester
Articles
Darnell Jr.,J.E.(1985) RNA.Scientific American,253,5472
Felsenfeld,G.(1985) DNA.Scientific American253,5472
The Molecular Basis of Evolution.(1985) Scientific American253,148157
Weinberg,R.A.(1985) The Molecules of Life.Scientific American253,3443
63
Teaching aids
Fletterick,R.J.,Schroer,T.and Matela,R.J.(1985) Blackwell Molecular Models(Patent No.437218)
together with Molecular Structure:Macromolecules in Three Dimensions,Blackwell Scientific
Publications,Oxford
Parish J.H.(1986) Nucleic Acid Structure and Synthesis,IRL Press Ltd.,Oxford
Bleasby,A.J.and Parish J.H.(1986) The Genetic Code and Protein Synthesis,IRL Press Ltd.,Oxford
Lowrie,R.S.Minit Molecular Modeling System.DNA and RNA model kits.Cochranes of Oxford
Ltd.,Oxford
Nicholson Molecular Models.DNA and RNA model kits.Labquip,Wokingham
The Bio-Bombshell (1984) New ScientistVideo,IPC Magazines Ltd.,London
The DNA Story,John Wiley and Sons Ltd.,Chichester
DNA - Blueprint of Life,John Wiley and Sons Ltd.,Chichester
Molecular Models Kit (DNA and RNA),Molecular Design Inc.,Academic Press,London
Pipe cleaners:useful for distinguishing left and right-handed helices
The Biochemical Basis of Biology(video programme),volumes 2 and 3,The Biochemical Society,
London
DNA Kit,NCBE,The University of Reading,Reading
Plant DNA Investigation Kit,NCBE,The University of Reading,Reading
DNA Model,NCBE,The University of Reading,Reading
64 Source materials
59
A form (crystalline form),of DNA, 13
Adaptor molecule, 18,41
Adenine, 7
Amino-acyl tRNA synthetase, 42,43
Anticodon, 44
Antiparallel arrangement,of DNA, 15
Bform, 13
Base composition
of DNA, 12,13
of RNA, 18
Base pairing, 12,14,17
Caesium chloride density gradient,
23,24
Central dogma of molecular biology,
29
Chain-terminating signal, 35
Chargaffs base pairing rule, 1214
Chemical taxonomy, 12
Chromatin, 11
Codon, 29,32
Condensation reaction, 10,44
Conservative replication, 22
Control region, 31
Crick,Francis, 14,32
Crystallographic repeat, 13
Cytosine, 7
Deformylase, 45
Degenerate code, 35
Density gradient centrifugation, 17,
23,24
Deoxyribonucleic acid (seeDNA)
Deoxyribonucleoside, 8
Deoxyribose, 6
DNA ligase, 27
DNA polymerase III, 26,27
DNA
A form (crystalline form), 13
antiparallel arrangement, 15
base composition, 12,13
duplex form, 16
isolation, 12
parallel arrangement, 15
replication, 27
structure, 1317,20,25
template, 37
Double helix, 14,25
Enzyme
in DNA replication, 26,27
in post-translational modification,
45
in transcription, 38
in translation, 42,43
Electrophoresis, 18
Elongation, 44
Epigenetic change, 2
N-Formylmethionine (fMET), 43
Gel filtration, 18
Gene region, 30
Genetic code, 29-31
Genetic information carrier, 2
Genotype, 2
Glycoside bond, 8
Guanine, 7
Heredity, 1,2
Histone protein, 11
Hybrid density, 24
Hydrogen bond, 1416,21,39
Identity period, 13
Initiation, 39
Initiation complex, 44
Initiation site, 40,43
Ion-exchange chromatography, 17
Isolation
of DNA, 12
of RNA, 17
Lagging strand, 26
Leading strand, 26
59
Subject index
Leder and Nirenberg, 33
Meselson and Stahl experiment,
23,24
Messenger RNA, 18,19,38,39
Minor base, 8
Multiple repeat sequence, 31
Non-coding sequence, 31
Nonsense codon, 34,45
Nucleoside, 8
Nucleotide, 8
Okazaki fragment, 26
One geneone polypeptide theory,
30
Palindromic region, 39
Parallel arrangement of DNA, 15
Peptidase, 45
Peptide bond, 44
Peptide synthesis, 3035,4345
Peptidyl transferase, 44
Perchloric acid, 12
Phenotype, 1,3
Phosphodiester linkage, 911
Polynucleotide, 9
Polysome/polyribosome, 46
Post-translational modification, 45
Protein (seePeptide synthesis)
Purine, 7
Pyrimidine, 7
Pyrophosphate linkage, 9
Replicase, 27
Replication, 2127
Replication fork, 26
Rho protein, 39
Ribonucleic acid (see RNA)
Ribonucleoside, 8
Ribose, 6
Ribosomal RNA, 18
Ribosome-binding technique, 33
Ribosome, 18,4346
Ribozyme, 20
Right-handed helix, 15
RNA polymerase, 3740
RNA
base composition, 18
isolation, 17
4S RNA, 19
structure, 1820
Semi-conservative replication, 22,25
Sequential code, 35
ShineDalgarno consensus sequence,
43
Single-stranded RNA, 16,20
Spent tRNA, 44
Strand separation, 26
Structure
of DNA, 1317,20,25
of RNA, 1820
Sugarphosphate backbone, 10,14,
16,17
Svedberg (S) unit, 19
Synthetic mRNA, 33
Termination, 39,45
Thymine, 7
Transcription, 29,31,3740
Transfer RNA, 18,19,44
Translation, 31,4146
Translocation, 44
Triple helix, 16
Triplet theory, 32
Uracil, 7
Virus, 12,16
Watson,James, 14
X-ray diffraction, 13,14
X174, 12,16
60 Subject index

Basc02 Full Dna

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The Structure and Function

*Department of Biological Sciences, Napier University, Edinburgh, and

Bolton School Boys Division, Bolton

Thi s process i s then repeated exactl y as above for a di fferent tri pl et

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