Chapter 9 For BB Lecture Notes 9 PDF

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Chapter 9 for BB - Lecture notes 9
Mendelian And Molecular Genetics (University of Illinois-Chicago)
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Chapter 9
•  Protein structure
•  The genetic code
•  Translation

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•  Genetic code
•  Translation

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Proteins are polymers (poly = many; mer = part) made

of amino acids attached end to end in a linear string.
The general formula for an amino acid is: H2N CHR
COOH in which the side chain, R, can be anything
from a hydrogen atom to a complex ring structure.
NH2-CH-COOH
R

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There are 20
common amino
acids found in
terrestrial life
forms, each with
unique chemical
properties that
are determined
by the R groups.

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Amino acids are connected by peptide bonds

between the carboxyl end of one and the amino end
of the next, thus forming a linear polypeptide chain.
Directionality!
Amino end Carboxyl end

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Proteins have four levels of structure

•  Primary (1˚) structure refers to the linear sequence of
amino acids in a polypeptide
•  Secondary (2˚) structure refers to interactions between
amino acids that are close together. 2˚ structure is usually
due to hydrogen bonding between the CO and NH groups
of nearby amino acid residues (e.g., α-helix and β-pleated
sheet).
•  Tertiary (3˚) structure refers to the 3D architecture of a
protein. It is determined by electrostatic, hydrogen, and
Van Der Waals bonds between R groups (often distant).
•  Quaternary (4˚) structure refers to the binding together of
two or more individual polypeptides to form a multimeric
protein (e.g., hemoglobin).

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Primary (1˚) structure — the sequence

of amino acids in a chain

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Secondary (2˚) structure — the first level of 3D

folding; determined by H-bonding between
nearby NH and CO groups
The two most

common types
of secondary
structure:
α-helix
and
β-pleated sheet

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Tertiary (3˚) structure

— the folding of the
secondary structures to
form the final 3D shape
of a polypeptide
The β chain of hemoglobin

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Some proteins possess Quaternary (4˚)

structure — several polypeptides
join together to form a multi-subunit structure
Hemoglobin is
made up of two α
and two β chains.

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Protein structure summary

Ø  Primary structure (the sequence of amino
acids) determines the secondary and
tertiary structures of the protein (the way
the string of amino acids folds in 3D space).
Ø  The amino acid side chains determine the
folding of the protein, and provide
functionality to the interaction surfaces and
to the active sites of enzymes.

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•  Genetic code
•  Translation

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Demonstration of the colinearity between gene & protein

In 1963, Charles Yanofsky demonstrated what was inferred from the
structure of DNA: there is a correspondence between the linear sequence of
a gene and its encoded polypeptide.
TrpA
TrpA
Gene: Mutations were mapped on DNA by P1 transduction (recomb. analysis).

Protein: The wild type and mutant proteins were sequenced.
Conclusion:
The linear sequence of nucleotides in a gene
determines the linear sequence of amino acids in its protein.

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Overview of the genetic code

•  The questions
–  Is the code overlapping or nonoverlapping?
–  How many letters (nucleotides) are there per word (amino acid)?
–  What is the code and what are its properties?
•  The tools
–  Logic and insight
–  DNA and protein sequencing
–  Synthetic RNA and programmed synthesis of polypeptides
–  Powerful genetic systems such as the rII locus of phage T4
–  tRNA conversion
–  mini mRNAs
•  The answers
–  The code is nonoverlapping and each codon has three letters.
–  All but three codons specify an amino acid; three codons specify stop.
–  The amino acids are illiterate
–  The code is read in a polarized fashion and is degenerate

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Is the genetic code overlapping or nonoverlapping?
reading 3 nts at a time
also reading 3 nts at a time

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How can we determine if the genetic code

is overlapping or nonoverlapping?
Experiment: Sequence mutant genes and their protein
products.
Ø  If the code is nonoverlapping, then a single base
change should alter only one amino acid in the
protein.
Ø  If the code is overlapping, then a single base
change should alter > 1 amino acid in the protein
(e.g., if 2 bases overlap, a single base change will
alter 3 adjacent amino acids).

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Overlapping or nonoverlapping?
Result: Mutation of a single base in a given gene
results in one amino acid change in corresponding
protein.
Conclusion: The genetic code is nonoverlapping !
This is true for a

given protein. It is
possible, although
rare, for two
different proteins
to be encoded by a
single mRNA
sequence read in
two different
frames.

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How many letters per word in

the genetic code?
Logic: There must be at least 20 words (codons) to encode
all 20 amino acids.
-  1 is not enough – only 4 codons (A, T, G, C)
-  2 is not enough – only 4 x 4 = 42 = 16 codons
-  3 is more than enough – 4 x 4 x 4 = 43 = 64!
Therefore, there must be at least 3 nts per codon.

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But is it 3 or is it more than 3?

In 1961, Francis Crick and Sidney Brenner
demonstrated that the genetic code uses three
letters (nucleotides) per word (amino acid).
Ø  Crick et al. used the rII locus of the T4 phage.
Ø  They began by mutagenizing the phage with
proflavin, which induces single base pair
insertions and deletions, and recovering rII
mutants. What is the phenotype of rII mutants?

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Crick’s and Brenner’s experiment

Goal: Isolate suppressors of mutations in the rII locus of T4
1. Induce rII mutations by proflavin, a mutagen that adds or
deletes single base pairs in DNA.
2. Mutagenize the rII mutants resulting from step 1 using the
same mutagen, and select for revertants that can grow
on E. coli K.
Wild type rII mutant Revertant

Growth on E. coli K: + - +
Revertants can be: true revertants or suppressors

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True Revertant
Example:
Code: ACT ACT ACT ACT ACT ACT
1st mutation:
insertion of G ACT GAC TAC TAC TAC TAC
True Revertant:
Deletion of the same G ACT ACT ACT ACT ACT ACT
Mutation wrong codons
Very rare

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Suppressor: a second mutation that counteracts the effects of

the first mutation. By definition, a suppressor mutation is at a
distinct site, and can be either intragenic or extragenic.
Example:
1st mutation:
2nd mutation:
Deletion of C ACT GAC TAT ACT ACT ACT
Mutation wrong codons

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Experiment: Suppressors of mutations in rII locus of T4:
Wild type rII mutant Revertant

Growth on E. coli K: + - +
Q: How can you distingusish a true revertant from a

suppressor?
A: If the second mutation does not restore the wild type

sequence, then the suppression of the mutant phenotype
must be due to a mutation at a second site, and the 2
mutations can be separated by recombination with wild
type.

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Double mutant
Mut #1 Mut #1 Suppressor
WT 2 single mutants
This is exactly what Crick and Brenner observed: second site mutations that
suppressed the rII phenotype conferred by the first mutation, but which caused
the same phenotype when isolated by recombination.

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Crick’s and Brenner’s experiment continued

•  In analyzing their data, Crick and Brenner found a striking
pattern:
v  Deletions (-) could suppress insertions (+).
v  Insertions (+) could suppress deletions (-).
v  However, changes of the same sign could not suppress one another,
with one important exception: triple mutations of the same sign (3
insertions or 3 deletions) often allowed wild type function!
•  Crick and Brenner concluded that the mRNA is read in a
polarized fashion and proceeds three nucleotides at a time.
Their observations can be explained as follows:
–  Insertion and deletion mutations alter the reading frame, and thus are
called frameshift mutations.
–  Suppressor mutations introduce a compensatory change.

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Frameshift mutations
The triplet code can be read in three possible frames

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Experiment: Suppressors of Mutations in rII locus of T4:
Mechanism: The triplet code (3 nucleotides/amino acid) is
read in a polarized fashion (i.e., in one direction), and can
be read in three possible frames.
First mutation: +/- out of reading frame,

or frameshift mutation
Second mutation: -/+ back to frame,
and sequence is back to correct, back to sense
In between: a short stretch of missense!
(cannot be too long, or the protein will be nonfunctional)

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Example:
1st mutation: frame-shift

2nd mutation: back to frame

Deletion of C ACT GAC TAT ACT ACT ACT
Mutation missense codons

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Why is the second mutation deleterious by itself?

Example:
2nd mutation by itself: frame-shift

Deletion of C ACT ACT ATA CTA CTA CTA
Both the 1st and the 2nd mutations are frameshift mutations!!

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Observation: Triple mutations of the same sign (+/+/

+ or -/-/-) can restore wild type function
Example:
Code: ACT ACT ACT ACT ACT ACT….
3 deletions: ACT ATC TCT ACT ACT…..

Back to frame
Conclusion: the code is exactly 3 nucleotides per codon

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The definitive evidence for polarized and

framed reading of the genetic code
George Steisinger used proflavin to induce mutations in the
gene that encodes lysozyme, a protein with a known amino
acid sequence.
Wild type -thr-lys-ser-pro-leu-asn-ala-
revertant -thr-lys-val-his-leu-met-ala-
Explain how this proves polarized and

framed reading of the code.
Question: Why do mutant alleles with intragenic suppressor

mutations often encode proteins with only partial function?
Question: How many reading frames are there for a given
molecule of DNA?

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A triplet code read in a single frame in a

polarized fashion
THEFATRATATETHEREDANTANDTHEBIGCOW
THE FAT RAT ATE THE RED ANT AND THE BIG COW
T HEF ATR ATA TET HER EDA NTA NDT HEB IGC OW
TH EFA TRA TAT ETH ERE DAN TAN DTH EBI GCO W
Crick
What andcan Brenner
we deduce could remove
if we removeor or
add one
add oneor
more
or more bases in the
letters rII gene
in this and ask
sentence and whether
ask it
still coded
whether for amakes
it still functional
sense ?
protein.

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How did Crick and Brenner infer that codons

are 3 nucleotides long?
THE
X FAT RAT ATE THE RED ANT AND THE BIG COW
H
THF ATR ATA TET HER EDA NTA NDT HEB IGC OW
THF ATR ATA HTE THE RED ANT AND THE BIG COW
•  So how did they deduce that the code uses 3 letters per
word?
•  Why does a suppressor mutation cause loss of function
when it is isolated?
•  Why does a mutant gene with a downstream suppressor
often only have partial function?

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Next property of the genetic code: degeneracy

•  The genetic code is degenerate, which is another way of
saying that it is redundant. This means that some amino
acids are specified by more than one codon.
•  Evidence for degeneracy of the code (Crick's supposition)
–  Hypothesis: The code is not degenerate. Each of the 20 amino acids
is specified by a single codon, and the other 44 codons specify no
amino acid—i.e., they are nonsense (termination) codons).
–  Predictions:
•  All frameshift mutations should result in immediate (or almost immediate)
termination of translation. Why?
•  Suppression of frameshift mutations will not occur because translation will
be terminated before a second site mutation can restore the reading
frame.
–  Observations:
•  Frame mutations usually do not result in immediate termination.
•  Frameshift suppressors arise frequently.
–  Conclusion: The code is degenerate

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Summary so far: The genetic code is

nonoverlapping, read in a polarized
fashion 3 nucleotides at a time, and is
degenerate.
But what is the code?

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Cracking the Code #1:

Random incorporation of nucleotides one at a time
•  The first breakthrough was the production of simple, defined
molecules of RNA.
–  In 1961, Nirenberg and Matthaei succeeded in synthesizing RNA
from scratch — i.e., in the absence of DNA — using the enzyme
polynucleotide phosphorylase and randomly incorporated
nucleotides.

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Cracking the Code #1:

Random incorporation of nucleotides one at a time
•  The first breakthrough was the production of simple,
defined molecules of RNA.
–  In 1961, Nirenberg and Matthaei succeeded in synthesizing
RNA from scratch — i.e., in the absence of DNA — using the
enzyme polynucleotide phosphorylase and randomly
incorporated nucleotides.
–  Why couldn’t they have used RNA polymerase?
•  Synthetic RNAs were used to program the synthesis of
polypeptides in vitro using the translational machinery of
E. coli reconstituted from purified components.
•  The firt codon assignment was UUU. The
polynucleotide -UUUUUUUUUUUUUUU- encodes
polypeptide -Phe-Phe-Phe-Phe-.
•  The AAA, GGG, and CCC codons can be assigned in
the same way.
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What is the code?

The breakthrough was the ability to make RNA
without DNA:
Ribonucleotides (ATP, GTP, CTP, UTP) +
the enzyme polynucleotide phosphorylase
Single stranded RNA of random sequence

(No need for transcription of DNA)
This artificial RNA was then used to make the
encoded polypeptide(s) in vitro

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What is the code?
Synthetic RNA:
PNP + UTP (uracil triphosphate)
UUUUUUUUUUU …, or Poly(U)
Poly (U) + protein synthesizing machinery
Protein: Poly-phenylalanine
Phe-Phe-Phe-Phe-Phe-Phe …
Nirenberg
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What is the code?
Conclusion:
Codon aa
UUU phenylalanine
Later added: AAA, CCC, GGG
CCC proline
AAA lysine
GGG glycine

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Cracking the Code #2: Repeating nucleotide polymers

•  In this approach, a repeating nucleotide polymer such as
(AGA)n is used to program in vitro translation ---> the
sequences of the resulting polypeptides are determined.
•  The predicted codons are then correlated with the resulting
proteins.
•  Unambiguous assignments can only be made by repeating the
experiment with a large number of nucleotide polymers. (See
prob. 44 — note that the first line should be (UC)n not (UG)n
•  H. Khorana finished breaking the code with this method and
also won a Nobel prize.
•  What’s the difference between this method and that used by
Nirenberg and Matthaei?
–  In the Nirenberg method, only one nucleotide was used.
–  In the Khorana method, two and three different nucleotides were used
to generate repeating sequences. Different polypeptides are produced
because different reading frames are used.

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The repeating nucleotide polymer approach

Synthetic RNA:
Clever chemistry allowed synthesis of short RNA
oligonucleotides of known sequence
PNP + CU
(CU)n, or CUCUCUCUCUCUCU …
Two possible frames here: UCU-CUC-UCU-CUC

or CUC-UCU-CUC-UCU
In both cases, the protein produced has alternating
serine and leucine: Ser-Leu-Ser-Leu-Ser-Leu …
Khorana (problem 44)
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Some synthetic RNAs failed to produce any protein:
For example, (GAUA)n and (GUAA)n
The reason – three codons do not encode any amino acids.

These are UAA, UAG, and UGA, stop-codons
GAUAGAUAGAUAGAUA
GUAAGUAAGUAAGUAA
Khorana (problem 44)

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Cracking the Code #3: Identification of the Stop codons

•  How Brenner deduced the role for UAG continued
1.  Brenner isolated 6 mutants of the T4 phage that had shortened head
proteins.
2.  The mutant proteins were sequenced.
3.  By comparing the sequences of the mutant and wild type proteins,
Brenner determined the identity of the amino acid that would have
been inserted next in each of the mutants, had the mutations not
occurred.
4.  In thinking about these six amino acids — glutamine, lysine, glutamic
acid, tyrosine, and serine — Brenner deduced that each was specified
by a codon that could mutate to UAG in a single step. Why is it
important that UAG could be produced in a single step?
5.  For each of his six head mutants, Brenner isolated suppressor
mutations in the host chromosome that restored wild type head length.
How might these suppressors work?
•  5'-UAC-3' codes for tyrosine, so it's anticodon is 3'-AUG-5'. A G --> C
mutation will yield 3'-AUC-5' which recognizes the 5'-UAG-3' Stop
codon.
•  Question: Is this an intragenic or extragenic suppressor?
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Cracking the Code #4: mini RNAs
•  The code was fully deciphered using mini RNAs, and

repeating nucleotide polymers.
•  Mini RNAs are three nucleotides in length, of known
sequence.
•  The amino-acyltRNA that a given mini RNA binds in vitro is
dictated by the code.
–  Example: GUU binds valyl-tRNA in vitro. Therefore, GUU must
code for valine.
–  Note that this type of experiment alone could have been used to
decipher the code. Why wasn’t it?

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How is the nucleic acid triplet code

translated into a sequence of amino acids?
Could the mRNA fold in such a way as to
specify the amino acid sequence directly?
•  Not likely. If mRNA is mixed with all 20 amino acids in a

test tube, protein is not synthesized. What else is
required?
•  In 1958, Crick brilliantly hypothesized the existence of
adapter molecules that fit the amino acids to the mRNA
template, with the order of amino acids determined by
nucleotide base pairing between the adapter and template.
•  Crick speculated further that a distinct enzyme would be
required to attach each adapter to its own amino acid.

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The adaptor is
tRNA, which
binds the amino
acid to the codon
and ribosome.
Improper base
pair?
• The 2˚ structure looks
like a cloverleaf.
• Note that the tRNA
molecule has two
"business ends."
• Note that the anticodon
is complementary and
antiparallel to the
codon.
Modified nucleotides Anticodon 5' to 3'

found only in tRNA:
ψ, UH2, and mI (there
are others)

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The 3D structure of the various tRNAs

is very similar, but not identical
phenylalanine
glutamine
Unique shapes at
the amino acyl
end and the
anticodon loop

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Aminoacyl-tRNA synthesis joins each

amino acid with its tRNA(s)

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Aminoacyl-tRNA synthesis
aax + tRNAx + ATP aax-tRNAx + AMP + PPi
Ø  This reaction is catalyzed by the enzyme amnoacyl-
tRNA synthetase. There are 20 such enzymes—1 for
each amino acid.
Ø  Each aminoacyl-tRNA synthetase recognizes a particular
amino acid and attaches it to all tRNAs that carry that
amino acid.
Ø  Therefore, the accuracy (specificity) of protein synthesis
depends on the ability of the enzyme to distinguish its
particular amino acid and set of corresponding tRNAs
from all other amino acids and tRNA species.

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Evidence that aminoacyl-tRNA synthesis

provides the specificity of translation
Experiment: chemically change the amino acid on a tRNA
to another amino acid
cysteine-tRNACys -------------> alanine-tRNACys
Synthesize protein with alanine-tRNACys
Result: Protein with alanines where the cysteines should be.
Conclusion: The tRNA recognizes the codon, not the amino

acid. The aminoacyl tRNA synthetases provides the
specificity, not the ribosome.
By joining the correct amino acids and tRNAs, the
synthetases do the actual translation of the code!

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Degeneracy Revisited
•  The genetic code is degenerate, which is another way of
saying that it is redundant. This means that some amino
acids are specified by more than one codon.
How is this achieved?
•  There are two distinct sources of degeneracy (redundancy)
in the code:
1. Some aminoacyl tRNA synthetases couple their
particular amino acid to more than one species of tRNA.
In other words, tRNA molecules with different anticodons
can carry the same amino acid.
2. In some cases, a given species of tRNA recognizes more
than one codon, due to wobble.

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Wobble: An odd inexactitude
: In the 3rd position, G, U,

and I can assume either of 2
positions, and thus can form
unusual base pairs. Recall
that H-bonds are highly
directional.
•  Some tRNAs recognize more than one codon! Sloppy

pairing (wobble) at the 3rd base of a codon allows one
tRNA to recognize two or even three different codons.
•  Notice that most codons for the same amino acid differ in
the third nucleotide.

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Most codons for the same amino acid

differ in the third nucleotide
Serine, leucince,
and arginine are
encoded by six
codons.

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Rules of
Wobble
pairing
Serine codons: UCU, UCC, UCA, UCG, AGU and AGC
tRNASer1 3'-AGG-5'
anticodon pairs
with both UCU
and UCC:

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Properties of the Genetic Code

Summary
The genetic code is nonoverlapping, uses
three letter words that are read in a
polarized fashion (in one direction),
depends directly on tRNAs and the tRNA
synthetases for specificity, and is highly
degenerate (redundant).

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THE CODE

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•  Genetic code
•  Translation

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Protein synthesis: directed polymerization

of amino acids
•  What is a polymer? Polymers are large
molecules composed of repeating structural
units.
•  What are some examples of polymers in
biological systems?
–  RNA & DNA
–  Polypeptides (proteins)
–  Glycogen
–  Fats
•  Why is protein synthesis referred to as directed
polymerization?

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Getting ready to polymerize

These requirements
In order arewe
to polymerize, satisfied
need: by . . .
•  a mechanisnm for linking the subunits together

the ribosome
•  a mechanism(s) that provides specificity
the tRNA—aminoacyl tRNA synthetase interaction
(and the anticodon)
•  energy to drive the reaction forward and thus
allow a decrease in entropy
ATP

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Charging the tRNA: a prelude to

protein synthesis
Charging the tRNA means to attach the aa to it.
aax + tRNAx + ATP + aminoacyl-tRNA synthetasex
aax-tRNAx + AMP + PPi
The energy provided by ATP is stored

in the aa-tRNA bond

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Peptide bond formation (polymerization)
aa1-tRNA1 + aa2-tRNA2
aa1- aa2-tRNA2 + tRNA1
Catalyzed by peptidyl transferase and

the ribosome
No additional energy required!

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Dissection of the translational machinery

•  The components of the translational machinery were
identified by centrifugation through sucrose density
gradients.
•  This method separates molecules by the rate at
which they move through the gradient, and thus
according to their size and shape.
–  The largest molecules reach the bottom of the tube first.
These have the larger S value. (S stands for
sedimentation coefficient.)
–  Equilibrium is not reached.
•  In contrast, centrifugation through CsCl density
gradients separates molecules according to their
density (see chapter 7). Molecules band where they
are as dense as the gradient. Equilibrium is
reached.
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Dissection of the translational machinery

•  The translational machinery consists of several
classes of macromolecule.
–  tRNA (transfer RNA)
–  mRNA (messenger RNA)
–  Ribosomes
•  Protein
•  rRNA: 23S, 16S, 5S (ribosomal RNA)
•  You should know of these various components,
but don't memorize details such as their exact
sizes.
•  tRNA and rRNA molecules are encoded by
what?

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Ribosome
Two subunits:
large and small
Both consist of
RNA molecules
and many protein
molecules.
About 2/3 of the
ribosome’s mass
is RNA.

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Ribosome
rRNA molecules are
huge and complex.
The 16S prokaryotic
rRNA from the small
subunit of the
ribosome is >1500
nucleotides long.

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Q: Why doesn't 50S + 30S = 80S and 60S + 40S = 100?

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What you should know about the structure of ribosomes

•  All ribosomes are composed of a large and a small subunit.
–  The eukaryotic ribosomal subunits and assembled ribosome are
slightly larger than their prokaryotic counterparts.
–  The ribosomal subunits remain apart until the initiation of
translation.
•  The ribosomal subunits are composed of both protein and
RNA.
–  Each subunit contains a large ribosomal RNA species. Thus, there
are two large rRNAs in each ribosome.
–  The large eukaryotic subunit contains two additional small rRNA
species, whereas the large prokaryotic subunit only contains one.
–  All ribosomal RNAs and proteins are encoded by genes.
–  The ribosomal proteins are thought to be structural and the
ribosomal RNAs are thought to be catalytic!

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What’s the goal here?
peptidyl aminoacyl Adapter
The ribosome reads the message in the 5' --> 3' direction. In what
direction is the gene read?

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Amino acids are connected by peptide bonds

between the carboxyl end of one and the amino end
of the next, thus forming a linear polypeptide chain.
Directionality!
Amino end Carboxyl end

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A site: entry of new Aminoacyl-tRNA

P site: growing Peptide-tRNA
(E Site: tRNA Exit)

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The three stages

Just like transcription, translation can
be divided into three stages:
Ø  INITIATION
Ø  ELONGATION
Ø  TERMINATION
Not to be confused with the three stooges: Larry, Moe, and Curly

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Initiation of translation — key points

•  Two things are accomplished during initiation.
–  The ribosome is assembled in a complex containing
the mRNA and the first tRNA.
–  The ribosome finds the initiation position (a particular
AUG or GUG)
•  Initiation occurs in 3 steps, requiring 3 factors.
–  The the small subunit of the ribosome binds to the
mRNA. This step requires IF3 (initiation factor 3).
–  N-formylmethionine binds to the P site of the
ribosome. This step requires IF2-GTP.
–  The large subunit binds to the complex, thus
completing assembly. The energy for this reaction is
derived from the hydrolysis of the GTP bound to IF2.

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Translation initiation in prokaryotes
Initiation Factors
IF1, IF2, and IF3
help the initiator
tRNA position
properly, then are
replaced by the
large subunit.

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Differences between initiation of translation

in prokaryotes and eukaryotes
•  Prokaryotes use fMet for initiation and methionine
thereafter; eukaryotes use only methionine.
•  Prokaryotes use AUG, GUG, and UUG as
initiation codons; eukaryotes use only AUG.
•  Prokaryotic messages are translated as they are
being transcribed; eukaryotic messages must first
be processed and transported to the cytoplasm.
–  Prokaryotic messages are polycistronic (polygenic).
Therefore, prokaryotic ribosome will have to find more
than one initiation codon on each mRNA.
–  Eukaryotic messages are monocistronic (monogenic),
but have leader sequences and poly A tails.

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Polygenic vs. monogenic messages
prokaryotic
eukaryotic
Moreover, there are multiple occurrences

of AUG in any given message.
How does the ribosome find the right AUG (and
therefore the correct frame) to initiate translation?
Prokaryotes and eukaryotes use different mechanisms.

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Specificity of initiation:
How does the ribosome know where to start?
•  John Shine and Lynn Dalgarno noticed that the true initiation
codons are preceded by sequences that are complementary to
the 3' end of the 16S rRNA. The interaction between this
Shine and Dalgarno sequence and the 16S rRNA helps
position the ribosome for proper initiation.
Interaction between the

S&D sequence and the
3' end of the 16S rRNA.

lOMoARcPSD|693378
Translation initiation in prokaryotes
Initiation Factors
IF1, IF2, and IF3
help the initiator
tRNA position
properly, then are
replaced by the
large subunit.

lOMoARcPSD|693378
Translation initiation in eukaryotes

Initiation factors
eIF4A, B, C bind to
the 5'-Cap on the
mRNA and recruit
the small (40S)
subunit. Together
with initiator tRNA,
this complex scans
the mRNA sequence
for the presence of
AUG (START)
codon.

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Translation initiation in eukaryotes continued.
Then eIF4A, eIF4B, and

eIF4C are replaced by
+ GTP
heavy (60S) subunit of the
ribosome.

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Translation elongation
•  Similar in prokaryotes and eukaryotes.
•  Requires several Elongation Factors:
EF-Tu, EF-Ts, and EF-G.
•  In addition to the ATP used by the
aminoacyl-tRNA synthetases, elongation
requires about 3 more GTP molecules
for each added amino acid.

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tRNA path:
A→P→E
Peptide path:
P→A→P→A

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Translation termination
1
•  The stop codons UAG, UGA, and UAA
are not recognized by tRNAs, but by
proteins called release factors.
•  When the ribosome encounters a stop
codon and the peptidyl-tRNA is in the P
site (1), the release factors bind to the A 2
site (2). This causes the polypeptide to
be released and the ribosome to
dissociate into its two subunits in a
reaction driven GTP hydrolysis (3). 3
•  The termination mechanism is similar in
prokaryotes and eukaryotes.
•  tRNAs with mutated anticodons can act
as nonsense suppressors (see Fig.
9.18), but an abundance of such tRNAs
can be detrimental to the cell. Why?

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Protein Processing
Many proteins are posttranslationally modified
•  Phosphorylation – regulation of activity. Kinases
add phosphates and phosphatases remove
phosphates.
•  Addition of lipids – helps proteins bind to the
membrane
•  Addition of short peptides (e.g., ubiquitin) –
triggers degradation of proteins.
•  Glycosylation – important for proteins on the cell
surface (like blood group antigens)
•  Splicing of proteins has been reported as well.
•  Many other examples are known.

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Protein targeting examples
•  Secreted proteins or cell surface proteins are

synthesized on the Endoplasmic Reticulum and
sorted through the Golgi.
•  Nuclear proteins have a special short amino-acid
sequence called the NLS (Nuclear Localization
Sequence) that triggers their transport to the
nucleus.
•  Most mitochondrial proteins are synthesized in
mitochondria.

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Protein targeting
Proteins that are needed
synthesized on the
Endoplasmic Reticulum

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Gene structure diagram

Promoter:
-35 -10
ATG Terminator
+1 start (start codon) stop codon
Translation

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Universality of the genetic code

Information transfer is the same in all organisms:
Replication DNA --> DNA
Transcription DNA --> RNA
Translation RNA --> protein
RNA from any organism can be transcribed
and translated in any other organism to give
protein with the same sequence!
Conclusion: the genetic code and information

transfer mechanisms are universal!

lOMoARcPSD|693378
End of chapter 9

Chapter 9 For BB Lecture Notes 9 PDF

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Chapter 9 for BB - Lecture notes 9

Mendelian And Molecular Genetics (University of Illinois-Chicago)

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Proteins are polymers (poly = many; mer = part) made

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Amino acids are connected by peptide bonds

Amino end Carboxyl end

Proteins have four levels of structure

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Primary (1˚) structure — the sequence

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Secondary (2˚) structure — the first level of 3D

The two most

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Tertiary (3˚) structure

The β chain of hemoglobin

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Some proteins possess Quaternary (4˚)

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Protein structure summary

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Demonstration of the colinearity between gene & protein

Gene: Mutations were mapped on DNA by P1 transduction (recomb. analysis).

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Overview of the genetic code

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Is the genetic code overlapping or nonoverlapping?

reading 3 nts at a time

also reading 3 nts at a time

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How can we determine if the genetic code

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Conclusion: The genetic code is nonoverlapping !

This is true for a

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How many letters per word in

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But is it 3 or is it more than 3?

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Crick’s and Brenner’s experiment

Wild type rII mutant Revertant

Revertants can be: true revertants or suppressors

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Mutation wrong codons

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Suppressor: a second mutation that counteracts the effects of

Mutation wrong codons

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Experiment: Suppressors of mutations in rII locus of T4:

Wild type rII mutant Revertant

Q: How can you distingusish a true revertant from a

A: If the second mutation does not restore the wild type

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Crick’s and Brenner’s experiment continued

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First mutation: +/- out of reading frame,

•  The code was fully deciphered using mini RNAs, and

•  Not likely. If mRNA is mixed with all 20 amino acids in a