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Nucleic Acids

• DNA (Deoxyribonucleic Acid)

NUCLEIC ACIDS • RNA (Ribonucleic Acid)

Nucleic Acids Pyrimidine/Purine Bases

• Nucleic acid: a biopolymer containing three types of • The structures of pyrimidine and purine:
monomer units

• a sugar (a pentose), either

D-ribose or 2-deoxy-D-ribose

• a base derived from purine

or pyrimidine (nucleobases)

• phosphate

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Other Bases
• Less common bases
can occur
• Principally but not
exclusively, in transfer
RNAs
Nucleotides
• Nucleotide: PO4 + sugar +
base
• a nucleoside + phosphoric acid
• phosphoester bond with an -OH
of the sugar, most commonly
either the 3’-OH or the 5’-OH
• Name based on parent
nucleoside with a suffix
“monophosphate”
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Nucleosides
• Nucleoside: sugar + base
• D-ribose or 2-deoxy-D-ribose covalently bonded to a
nucleobase by a  -N-glycosidic bond
• Lacks phosphate group
Nucleic Acids
• Polymerization of
nucleotides leads to
nucleic acids.
• Linkage is repeated
• (3’,5’-phosphodiester
bond)
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Nucleic Acids DNA - 1° Structure

• Levels of structure
Deoxyribonucleic acids, DNA:
• 1°structure: the order of bases on the
polynucleotide sequence; the order of bases • Primary Structure: the sequence of bases along the sugar-
specifies the genetic code phosphodiester backbone of a DNA molecule
• 2°structure: the three-dimensional conformation • base sequence is read from the 5’ end to the 3’ end
of the polynucleotide backbone • System of notation single letter (A, G, C and T)
• 3°structure: supercoiling
• 4°structure: interaction between DNA and A
3’
C G T G
O 3’
or 5’ P
A C G T G
O 3’
H
proteins P P P P H

5’
P
5’

DNA - 2° Structure T-A Base Pairing

• Secondary structure: the • Base pairing is complementary
ordered arrangement of
nucleic acid strands
• the double helix model • A major factor stabilizing the double helix is base pairing by
of DNA 2°structure was hydrogen bonding between T-A and between C-G
proposed by James
Watson and Francis • T-A base pair comprised of 2 hydrogen bonds
Crick in 1953
• Double helix:
• two antiparallel
polynucleotide strands are
coiled in a right-handed
manner about the same
axis
• structure based on X-Ray
crystallography

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G-C Base Pair Forms of DNA 2o Structure

• B-DNA
• G-C base pair comprised of 3 hydrogen bonds •considered the physiological form
•a right-handed helix, diameter
11Å
•10 base pairs per turn (34Å) of
the helix
•occurs in nature
• A-DNA
•a right-handed helix, but thicker
than B-DNA
•11 base pairs per turn of the helix
•has not been found in vivo
• Z-DNA
• a left-handed double helix
• may play a role in gene
expression

Other Features of DNA DNA – 3o Structure in Prokaryotes

• Base stacking Tertiary structure: the 3-D arrangement
• bases are hydrophobic and of all atoms of a nucleic acid;
interact by hydrophobic
interactions • commonly referred to as supercoiling
• in standard B-DNA, each
base rotated by 32°
• Circular DNA: a type of double-
compared to the next stranded DNA in which the 5’ and 3’
ends of each strand are joined by
phosphodiester bonds
• Supercoiling- Further coiling and
twisting of DNA helix.

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DNA – 3o Structure in Prokaryotes Super DNA Coiled Topology

• Supercoiling- Further coiling and twisting of • Double helix can be considered to be a 2-stranded,
DNA helix. right handed coiled rope
• Can undergo positive/negative supercoiling
• Topoisomerases- an enzyme that relaxes
supercoiling in closed circular DNA
• Class I: cut the phosphodiester backbone
of one strand, pass the end through, and
reseal
• Class II: cut both strands, pass some of
the remaining DNA helix between the cut
strands, and reseal
• DNA gyrase: a bacterial topoisomerase II

Supercoiling (3o Structure) in Eukaryotic DNA Denaturation of DNA

• Histone: a protein, particularly rich in
the basic amino acids Lys and Arg;
found associated with eukaryotic DNA
• five main types: H1, H2A, H2B, H3,
H4
• Denaturation: disruption of 2o structure
• most commonly by heat denaturation (melting)
• double helix unwinds when DNA is denatured
• Renaturation
• double helix can be re-formed with slow cooling and annealing

• Chromatin: DNA molecules wound

around particles of histones in a
beadlike structure
• Each “Bead” is a nucleosome
• Nucleosome consists of: DNA
wrapped around histone core

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DNA REPLICATION
Prokaryotic DNA Replication
• Replication involves
• separation of the two original
strands
• synthesis of two new
daughter strands using the
original strands as templates
• Semiconservative
replication: each daughter
strand contains one template
strand and one newly
synthesized strand
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Bidirectional Replication
Which Direction does Replication go?
• DNA double helix unwinds at a specific point called
an origin of replication
• DNA replication is bidirectional in most organisms;
Polynucleotide chains are synthesized in both
directions from the origin of replication
• At each origin of replication, there are two
replication forks, points at which new polynucleotide
chains are formed
• There is one origin of replication and two replication
forks in the circular DNA of prokaryotes
• In replication of a eukaryotic chromosome, there are
several origins of replication and two replication forks
at each origin
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Summary of DNA Replication in

Replication Fork General Features
Prokaryotes
• Unwinding
• DNA gyrase introduces a swivel point
in advance of the replication fork
• a helicase binds at the replication
fork and promotes unwinding
• single-stranded binding (SSB)
protein protects exposed regions of
single-stranded DNA
• Primase catalyzes the synthesis of RNA
primer
• Synthesis
• catalyzed by Pol III
• primer removed by Pol I
• DNA ligase seals remaining nicks

Replication Fork General Features

RNA

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RNA Flow of Genetic Information in the Cell

• Mechanisms by which information is transferred in
• RNA the cell is based on “Central Dogma”
• unbranched chains of nucleotides
• the sugar unit is -D-ribose
• Uracil instead of thymine
• in general, RNA is single stranded

A C G U G
3’ O 3’
or 5’ P
A C G U G
O 3’
H H
P P P P

5’
P
5’

Information Transfer in Cells

• Information encoded
in the nucleotide
sequence of DNA is
transcribed through
RNA synthesis

• RNA and amino acid

sequence then is
dictated by DNA
sequence

• Central dogma of
biology

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Transcription Transcription in Prokaryotes

• Overview of Transcription
• RNA is synthesized on a DNA template, catalyzed by
DNA-dependent RNA polymerase
• ATP, GTP, CTP, and UTP are required, as is Mg2+
• no RNA primer is required
• the RNA chain is synthesized in the 5’ -> 3’ direction;
the nucleotide at the 5’ end of the chain retains its
triphosphate (ppp) group
• RNA polymerase unwinds the helix
• the DNA base sequence contains signals for initiation
and termination of RNA synthesis; the enzyme binds
to and moves along the DNA template in the 3’ -> 5’
direction
• the DNA template is unchanged
• E. coli RNA Polymerase:
• four different types of subunits: ,  , ’, and s
• the core enzyme is 2’
• the holoenzyme is 2’s
• the role of the s subunit is recognition of the
promoter locus; the s subunit is released after
transcription begins
• of the two DNA strands, the one that serves as the
template for RNA synthesis is called the template
strand or antisense strand; the other is called the
coding (or nontemplate) strand or sense strand
• the holoenzyme binds to and transcribes only the
template strand

Parts of a gene The Basics of Transcription

PROKARYOTIC (Sense strand)

(Antisense strand)

Promoter Coding region Termination region

region

EUKARYOTIC

Promoter region

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Promoter Sequence Promoter Sequence

• Simplest of organisms contain a lot of DNA that is
not transcribed

• RNA polymerase needs to know which strand is

template strand, which part to transcribe, and where
first nucleotide of gene to be transcribed is

• Promoter – a DNA sequence that provides direction

for RNA polymerase

σ-subunit initiates strand separation (melting) of the DNA at about -10

from the start site.

Transcription Start Site

Promoter region
-10 +1
T G C T A G T C C T G C T A G C C G A T A T A A T G A C A A G A C G T C G A C T T A C A G C G-
---
A C G A T C A G G AC G A T C G G C T A T A T T A C T G T T C T G C A G C T G A A T G T C G C-
---

T G C TA G T C C T G C TA G C C G A T AT AA
T
A C G A T C A G G AC G A T C G G C T A T A T T
A

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Chain Elongation (Cont’d) Chain Termination

Two types of termination mechanisms:
(1) intrinsic termination- controlled by specific sequences,
termination sites
Termination sites characterized by two inverted repeats

Chain Termination (Cont’d) Eukaryotic mRNA Splicing

(2) Termination by rho () protein
• Rho-dependent termination sequences cause hairpin
loop to form

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Characteristic structure of eukaryotic mRNA RNA

• RNA molecules are classified according to their
structure and function

tRNA rRNA
amino acid
Transfer RNA, tRNA: Ribosomal RNA, rRNA:
• the smallest kind of the • found in ribosomes, the site of protein synthesis
three RNAs • only a few types of rRNA exist in cells
• a single-stranded • ribosomes consist of 60 to 65% rRNA and 35 to 40%
polynucleotide chain protein
between 73-94
• in both prokaryotes and eukaryotes, ribosomes
nucleotide residues
consist of two subunits: big and small subunits
• carries an amino acid at
• particles characterized by sedimentation coefficients,
its 3’ end
expressed in Svedberg units (S)
• intramolecular hydrogen • 40S and 60S subunits in eukaryotes
bonding occurs in tRNA
• 30S and 50S subunits in prokaryotes

anticodon

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mRNA snRNA
Messenger RNA, mRNA: • Small nuclear RNA (snRNA) is a recently
• carries coded genetic information from DNA to discovered RNA
ribosomes for the synthesis of proteins
• present in cells in relatively small amounts and very • Found in nucleus of eukaryotes
short-lived
• single stranded • Small (100-200 nucleotides long)
• biosynthesis is directed by information encoded on
DNA • Forms complexes with protein and form small
nuclear ribonucleoprotein particles (snRNPs)

• snRNPs help with processing of initial mRNA

transcribed from DNA

TRANSLATION
• The sequence of the bases on the mRNA
specifies the sequence of the amino acids in the
protein.
• Transcription takes place in the nucleus, then
the mRNA is transported to the cytosol
TRANSLATION: • Translation always takes place in the cytosol,
where the mRNA is read and translated at the
ribosome.
PROTEIN SYNTHESIS

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The Genetic code
• The sequence of amino
acids, derived from the
sequence of DNA bases,
is specified by the genetic
code, using 4 RNA bases
A, U, G, and C taken three
at a time (triplet code).
• There 64 possible “code
words”, called codons
• 1 start codon - AUG
• 3 are stop signals:
UAG, UGA, UAA
• 61 specify the 20
amino acids with
considerable
redundancy.
The Genetic Code (Cont’d)
• All 64 codons have
assigned meanings
•only Trp and Met have one
codon each
•the third base is irrelevant
for Leu, Val, Ser, Pro, Thr,
Ala, Gly, and Arg
•the second base is
important for the type of
amino acid; for example, if
the second base is U, the
amino acids coded for are
hydrophobic
•for the 15 amino acids
coded for by 2, 3, or 4
triplets, it is only the third
letter of the codon that
varies. Gly, for example, is
coded for by GGA, GGG,
GGC, and GGU
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The Genetic Code
• Salient features of the genetic
code
• triplet: a sequence of three
bases (a codon) is needed to
specify one amino acid
• nonoverlapping: no bases
are shared between
consecutive codons
• commaless: no intervening
bases between codons
• degenerate: more than one
triplet can code for the same
amino acid; Leu, Ser, and Arg,
for example, are each coded
for by six triplets
• universal: the same in
viruses, prokaryotes, and
eukaryotes; the only
exceptions are some codons in
mitochondria
Translating the Genetic Message
• Protein biosynthesis is
a complex process
requiring ribosomes,
mRNA, tRNA, and
protein factors
• Several steps are
involved
• Before being
incorporated into
growing protein chain,
a.a. must be activated
by tRNA and
aminoacyl-tRNA
synthetases
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Shine-Dalgarno Sequence Recognized by
E. Coli Ribosomes
The Initiation
Complex
• In all organisms,
synthesis of polypeptide
chain starts at the N-
terminal end, and grows
from N-terminus to C-
terminus
• Initiation requires:
• tRNA-fmet – binds to P
site
• initiation codon (AUG) of
mRNA
• 30S ribosomal subunit
• 50S ribosomal subunit
• initiation factors IF-1, IF-
2, and IF-3
• GTP, Mg2+
• Forms the initiation
complex
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Translation Start Site
+1
-10
T G C TA G T C C T G C TA G C C G A T AT AA
T
A C G A T C A G G AC G A T C G G C T A T A T T
A
A G C U G A A U G U C G C G C C U U U A C A G C …G A U G C C G U C G G U A G C U A A T U A
……
met - ser - arg - leu ……………………………………………… - ser
Elongation Steps
• Step 1
• an aminoacyl-tRNA is
bound to the A site
• the P site is already
occupied
• 2nd amino acid bound to
70S initiation complex;
defined by the mRNA
• Step 2
• EF-Tu is released in a
reaction requiring EF-Ts
• Step 3
• the peptide bond is
formed, the P site is
uncharged
• Step 4
• the uncharged tRNA is
released
• the peptidyl-tRNA is
translocated to the P site
• EF-G and GTP are
required
• the next aminoacyl-tRNA
occupies the empty A site
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UAG Simultaneous Protein Synthesis on

Chain Termination
Polysomes
• Chain termination requires
• stop codons (UAA, UAG,
or UGA) of mRNA
• RF-1 (Release factor-1)
which binds to UAA and
UAG or RF-2 (Release
factor-2) which binds to
UAA and UGA
• RF-3 which does not
bind to any termination
codon, but facilitates the
binding of RF-1 and RF-
2
• GTP which is bound to
RF-3
• The entire complex
dissociates setting free the
completed polypeptide, the
release factors, tRNA,
mRNA, and the 30S and
50S ribosomal subunits

Post-translational Modification Examples of Posttranslational Modification

• Newly synthesized polypeptides are frequently modified
before they reach their final form where they exhibit
biological activity
• N-formylmethionine in prokaryotes is cleaved
• specific bonds in precursors are cleaved, as for example,
preproinsulin to proinsulin to insulin
• leader sequences are removed by specific proteases of
the endoplasmic reticulum; the Golgi apparatus then directs
the finished protein to its final destination
• factors such as heme groups may be attached
• disulfide bonds may be formed
• amino acids may be modified, as for example, conversion
of proline to hydroxyproline
• other covalent modifications; e.g., addition of
carbohydrates

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2o Structure: the ordered 3-dimensional

Levels of Protein Structure arrangements (conformations) in localized regions of a
polypeptide chain; refers only to interactions of the
1° structure: the sequence of amino acids in a peptide backbone
polypeptide chain, read from the N-terminal end to
the C-terminal end
• 2° structure: the ordered 3-dimensional
arrangements (conformations) in localized regions of
a polypeptide chain; refers only to interactions of the
peptide backbone
• e. g., -helix and -pleated sheet
• 3˚ structure: 3-D arrangement of all atoms
• 4˚ structure: arrangement of monomer subunits
with respect to each other

α-Helix β-pleated sheet

3o Structure: 3-D arrangement of all atoms 4o Structure: arrangement of monomer subunits with
respect to each other
Forces That Stabilize Protein Structure
Structure of Hemoglobin

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MUTATION MUTAGENS
• Physical Agents
MUTATION - a change in the base sequence of DNA • UVL – pyrimidine dimers
Origins:
• Ionizing radiation – x-rays, gamma rays
Spontaneous mutation – occurs during normal
genetic and metabolic functions in the cell: • Chemical Agents
- Replication errors - genetic mispairing • Nitrous acid
- Base modifications caused by spontaneous • Intercalating agents
hydrolytic reactions • 5-bromouracil
- low frequency: 10-7 to 10-12/generation
Induced mutation – mutagens (increase frequency)

UVL Ionizing Radiation

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Chemical agents
MUTATION

• Addition
• Deletion
• Substitution
• Transition
• transversion

MUTATION Mutants

• Missense mutation • Missense mutant

• Nonsense mutation
• Base substitution in DNA causes replacement
• Silent mutation of 1 amino acid residue by another in a protein
• Frameshift mutation

AGU-CGU-GGA-AAU-UGU-CCU-CGA-
ser - arg - gly - asn - cys - pro - arg
AGU-CGU-GCA-AAU-UGU-CCU-CGA-
ser - arg - ala - asn - cys - pro - arg

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Mutants
• Nonsense mutant
• If base substitution creates a stop codon,
thereby terminates protein synthesis
prematurely

AGU-CGU-GGA-AAU-UGU-CCU-CGA-
ser - arg - gly - asn - cys - pro - arg
AGU-CGU-UGA-AAU-UGU-CCU-CGA-
ser - arg -stop

Mutants Mutants

· Silent mutant –
• Frameshift mutants
- substitution results in triplet coding for the same
amino acid as the original triplet (redundancy of • Due to insertion mutation or deletion mutation
the genetic code)
A
- change usually occurs at the 3rd base
AGU-CGU-GGA-AAU-UGU-CCU-CGA-
AGU-CGU-GGA-AAU-UGU-CCU-CGA-
ser - arg - gly - asn - cys - pro - arg -
ser - arg - gly - asn - cys - pro - arg
AGU-CGU-GAG-AAA-UUG-UCC-UCG-A-
AGU-CGU-GGU-AAU-UGU-CCU-CGA-
ser - arg - glu - lys - leu - ser - ser
ser - arg - gly - asn - cys - pro - arg

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REPAIR MECHANISMS LIGHT REPAIR

• Photoreactivation
• Excision repair
• Mismatch Repair

EXCISION REPAIR OR DARK REPAIR Mismatch Repair in Prokaryotes

• Mechanisms of mismatch repair encompass:

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