The Genetic Code

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Chapter 15 The Genetic Code
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This chapter mainly describes the classic
experiments that led to the elucidation of the
genetic code , and lays out the rules by which
the code is translated . The nucleotide sequence
information is based on a three letter code ,
while the protein sequence information is based
on twenty different amino acids . The code is
degenerate with two or more codons (in most cases
) specifying the same amino acid . There are also
specific codons that indicate where translation
should start and where it should stop .
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This chapter contains four parts
Topic one The code is degenerate Topic two
Three rules govern the genetic code Topic three
Suppressor mutations can reside
in the same or a different gene Topic four
The code is nearly universal
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The outline 1.At the
very heart of the Central Dogma is the concept of
information transfer from the linear sequence of
the four letter alphabet of the polynucleotide
chain into the 20-amino acid language of the
polypeptide chain . 2.Translation takes
place on ribosomes . 3.Translation is
mediated by special adaptor molecules known as
tRNAs .
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The total number of permutations of the
triplets is 64,a value well in excess of the
number of amino acids .Which of these triplet
codons are responsible for specifying which amino
acids , and what are the rules that govern their
use ? In this chapter , we discuss the nature and
underlying logic of the genetic code , how the
code was cracked ,and the effect of mutations
on the coding capacity of messenger RNA .
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Topic one THE CODON IS DEGENERATE
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Degeneracy (???????) The phenomenon of many
amino acids are specified by more than one codon
. Synonyms (?????) Codons specified the same
amino acid .
Table 15-1 The Genetic Code
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The features of the code
1 . 61 of the 64 possible triplets specify an
amino acid , with the remaining three triplets
being chain-terminating signals . 2 . When the
first two nucleotides are identical , the third
nucleotide can be either cytosine or uracil and
the codon will still code for the same amino acid
. Often , adenine and guanine are similarly
interchangeable . 3 . Not all degeneracy is
based on equivalence of the first two nucleotides
. ( Figure 15-1 )
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CUC
CUG
Figure 15-1 Codon-anticodon pairing of two tRNA
Leu molecules
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How there can be great variation in the AT/GC
ratios in the DNA of various organisms without
correspondingly large changes in the relative
proportion of amino acids in their proteins ?
This can be explained by codon degeneracy ,
especially the frequent third-place equivalence
of cytosine and uracil or guanine and adenine .
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1-1 Perceiving Order in the makeup of the
Code
1 . The code evolved in such a way as to
minimize the deleterious effects of mutations .
1 ) . Mutations in the first position of a codon
will often give a similar ( if not the same )
amino acid . 2 ) . Codons with pyrimidines in
the second position specify mostly hydrophobic
amino acids , whereas those with purines in the
second position correspond mostly to polar amino
acids . 3 ) .
If a codon suffers a transition mutation in the
third position , rarely will a different amino
acid be specified .
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2 . Whenever the first two positions of a
codon are both occupied by G or C , each of the
four nucleotides in the third position specifies
the same amino acid . 3 . Whenever the first
two positions of the codon are both occupied by A
or U , the identity of the third nucleotide does
make a difference .
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1-2 Wobble in the Anticodon
Why the opinion of that a specific tRNA
anticodon would exist for every codon is wrong ?
1 ) . Highly purified tRNA species of known
sequence could recognize several different codons
. 2 ) . An anticodon base was not
one of the 4 regular ones , but a fifth base ,
inosine .
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Wobble concept ( ???? ) The base
at the 5 end of the anticodon is not as
spatially confined as the other two , allowing it
to form hydrogen bonds with any of several bases
located at the 3 end of a codon . (This is
devised by Francis Crick in1966)
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The wobble rules
1 . Not all combinations are possible , with
pairing restricted to those shown in Table 15-2 .
2 . The pairings permitted by the wobble
rules are those that give ribose-ribose distances
close to that of the standard AU or GC base
pairs . 3 . The wobble rules do not permit
any single tRNA molecule to recognize four
different codons .
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Table 15-2 Pairing Combinations with the Wobble
Concept
Base in 5 Anticodon Base in 3 Codon
G U or C C
G A U
U A or G I
A, U, or C
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The ribose-ribose distances for the wobble pairs
are close to those of AU or GC base pairs
Figure 15-2 Wobble base pairing
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Why wobble is not seen in the first (5)
position of the code ?
Restriction of bases movements 1 ) . In
the three-dimensional structure of tRNA , the
three anticodon basesas well as the two
following (3) bases in the anticodon loopall
point in roughly the same direction (figure 15-3)
. 2 ) . The first (5) anticodon base is at
the end of the stack and is perhaps less
restricted in its movements than the other two
anticodon baseshence , wobble in the third
(3) position of codon .
3 ) .
Not only does the third (3) anticodon base
appear in the middle of the stack , but the
adjacent base is always a bulky modified purine
residue .
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The adjacent base is always a bulky modified
purine residue.
Figure 15-3 Structure of yeast tRNA(Phe)
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1-3 Three Codons Direct Chain Termination
The chain-termination codons , UAA , UAG ,
and UGA ,are read not by special tRNAs but by
specific proteins known as release factors.
Release factors enter the A site of the ribosome
and trigger hydrolysis of the peptidyl-tRNA
occupying the P site , resulting in the release
of the newly synthesized protein .
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1-4 How the Code Was Cracked
The identification of the codons for a given
amino acid would require
1 . Exact knowledge of the nucleotide
sequences of a gene .(while the then-current
methods were very primitive . ) 2 . The
corresponding amino acid order in its protein
product . (At that time , the elucidation
,although a laborious process , was already a
very practical one . )
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1-5 Stimulation of Amino Acid
Incorporation by Synthetic mRNAs
The extracts prepared from dells of E. coli ,
that were actively engaged in protein synthesis
,were capable of incorporating radioactively-label
ed amino acids into proteins .
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1) . Protein synthesis in these extracts
proceeded rapidly for several minutes and then
gradually came to a stop . 2) .
There was a corresponding loss of mRNA owing to
the action of degradative enzymes present in the
extract . However , the addition of fresh
mRNA to extracts that had stopped making protein
caused an immediate resumption of synthesis .
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How the RNA is synthesized? XMPn XDP
XMPn1 P
Polynucleotide phosphorylase is normally
responsible for breaking down RNA , and no
template DNA or RNA is required for RNA synthesis
with this enzyme the product depends entirely
on the ratio of the various ribonucleoside
diphosphates added to the reaction mixture .
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1.Under physiological conditions favors the
degradation of RNA into nucleoside diphosphates .
2.By use of high nucleoside diphosphate
concentrations , this enzyme can be made to
catalyze the formation of internucleotide 3
5 phosphodiester bonds and thus make RNA
molecules (figure 15-4) . 3.In all these
mixed polymers ,the base sequences are
approximately random ,with the nearest-neighbor
frequencies determined solely by the relative
concentrations of the reactants .
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Figure 15-4 Polynucleotide phosphorylase reaction
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1-6 Poly-U Codes for Polyphenylalanine
1.Under the right conditions in vitro , almost
all synthetic polymers will attach to ribosomes
and functions as templates . 2.A high
magnesium concentration can circumvent the need
for initiation factors and the special initiator
fMet-tRNA , allowing chain initiation to take
place without the proper signals in the mRNA .
3.Pol-U selects phenylalanyl tRNA molecules
exclusively , thereby forming a polypeptide chain
containing only phenylalanine (polyphenylalanine)
.
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1-7 Mixed Copolymers Allowed Additional
Codon Assignments
We can ratiocinate the codon assignments by
the proportions of different codons .(the
proportions of the codons vary with the copolymer
base ratio , eg table 15-3 in page 468 . )
But there is no way of knowing the order from
random copolymers .
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1-8 Transfer RNA Binding to Defined
Trinucleotide Codons
Trinucleotide effect (??????) 1.Even in the
absence of all the factors required for protein
synthesis , specific aminoacyl-tRNA molecules can
bind to ribosome-mRNA complexes . 2.This
specific binding does not demand the presence of
long mRNA molecules . In fact , the binding of a
trinucleotide to a ribosome is sufficient .
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The function of the discovery of thes
trinucleotide effect It provided a
relatively easy way of determining the order of
nucleotides within many codons .
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1-9 Codon Assignments from Repeating
Copolymers
Organic chemical and enzymatictechniques were
being used to prepare synthetic
polyribonucleotides with known repeating
sequences (Figure 15-5) .
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Figure 15-5 Preparing oligo-ribonucleotides
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Ribosomes start protein synthesis at random
points along these regular copolymers yet they
incorporate specific amino acids into
polypeptides . (Table 15-5)
The sum of all these observations they
permitted the assignments of specific amino acids
to 61 out of the possible 64 codons ,with the
remaining three chain-terminating codons , UAG ,
UAA , AND UGA , not specifying any amino acid .
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Table 15-5
Amino Acids Incorporated or Polypeptide Made
Codons Recognized
Codon Assignment
copolymer
(CU) CUCUCUCUC Leucine
5-CUC-3
Serine UCU (UG)
UGUGUGUGU Cystine UGU

Valine GUG (AC)
ACACACACA Threonine ACA

Histidine CAC (AG)
AGAGAGAGA Arginine AGA

Glutamine GAG (AUC) AUCAUCAUC
Polyisoleucine 5-AUC-3

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Topic two THREE RULES GOVERN THE
GENETIC CODE
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The genetic code is subject to three rules that
govern the arrangement and use of codons in
messenger RNA
1 . Codons are read in a 5 to 3 direction .
(eg The base order of the dipeptide
NH2-Thr-Arg-COOH is 5- ACGCGA-3 ,but not
3-GCAAGC-5) 2 . Codons are nonoverlapping
and the message contains no gaps . 3 . The
message is translated in a fixed reading frame ,
which is set by the initiation codon .
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2-1 Three Kinds of Point Mutations Alter
the Genetic Code
1 . Missense mutation (????) An alteration
that changes a codon specific for one amino acid
to a codon specific for another amino acid .
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2 . Nonsense (????) or Stop mutation (????)
An alteration causing a change to a
chain-termination codon . (a more drastic effect
) 3 . Frameshift mutation (????)
Insertions or deletions of one or a small number
of base pairs that alter the reading frame .
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1 ) . The insertion (or for that matter the
deletion ) of a single base or two bases
drastically alters the coding capacity of the
message not only at the site of the insertion but
for the remainder of the messenger as well . 2
) . An insertion of three extra bases at nearby
positions in a message will drastically alter the
stretch of message , at and between the three
insertions . But mRNA downstream of the three
inserted bases will be in its proper reading
frame and hence , completely unaltered .
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eg Ala Ala Ala Ala Ala
Ala Ala Ala
5-GCU GCU GCU GCU GCU GCU GCU GCU-3
Insertion of an A in the message Ala
Ala Ser Cys Cys Cys Cys Cys
5-GCU GCU AGC UGC UGC UGC UGC
UGC-3 Insertion of a three extra bases at
nearby positions in a message Ala Ala Ser
Cys Met Leu His Ala Ala Ala
5-GCU GCU AGC UGC AUG CUG CAU GCU GCU
GCU-3
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2-2 Genetic Proof that the Code Is Read
in Units of Three
Francis Crick ?Sydney Brenner
a classic experiment involving
bacteriophage T4 Conclusion because the gene
could tolerate three insertions but not one or
two , the genetic code must be read in units of
three . (This did so purely on the basis of a
genetic argument . )
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Topic three SUPPRESSOR
MUTATIONS CAN RESIDE IN THE SAME OR A
DIFFERENT GENE
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The effects of harmful mutations can be reversed
by a second genetic change 1 . Simple reverse
(back) mutations (????) Mutations change
an altered nucleotide sequence back to its
original arrangement . 2 . Suppressor mutations
( ????) The mutations occurring at
different locations on the chromosome the
suppress the change due to a mutation at site A
by producing an additional genetic change at site
B .
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1 ) . Intragenic suppressor (?????) The
suppressor mutations occurring within the same
gene as the original mutation but at a different
site in this gene . ( such as missense mutation
and frameshift mutation . ) 2 ) . Intergenic
suppressor (?????) The suppressor mutations
occurring in another gene .
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Figure 15-6 Suppression of frameshift mutations
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3-1 Intergenic Suppression Involves
Mutant tRNAs
Suppressor genes do not act by changing the
nucleotide sequence of a mutant gene . (eg
figure 15-7) Cell with nonsense suppressors
contain mutationally-altered rRNAs Question
How their codons corresponding to these rRNAs
could continue to be read normally ?
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Figure 15-7 a Nosense suppression
Figure 15-7 a
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Figure 15-7 b Nosense suppression
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3-2 Nonsense Suppressors also Read
Normal Termination Signals
The act of nonsense suppression can be viewed
as a competition between the suppressor tRNA and
the release factor .
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1 .Suppression of UAG codons is efficient . In
the presence of the suppressor tRNA , more than
half of the chain-terminating signals are read as
specific amino acid codons . 2 . Mutant
cells producing UAA-suppressing tRNAs grow poorly
.
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E. Coli can tolerate this misreading of the
UAG stop codon , in contrast , suppression of the
UAA codon usually averages between 1and 5 , why
? Because UAG is used infrequently as a
chain-terminating codon at the end of
open-reading frames , while UAA is frequently
used as a chain-termination codon and its
recognition by a suppressor tRNA would be
expected to result in the production of many more
aberrantly long polypeptides .
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3-3 Proving the Validity of the Genetic
Code
The code was cracked , as we have seen , by
means of biochemical methods involving the use of
cell-free systems for carrying ort protein
synthesis . But how do we know definitively that
the code as depicted in Table is true in living
cells ? 1966 , An experiment was based on the
construction by genetic recombination of a mutant
gene of phage T4 that harbored a mutually
suppressing pair of insertion and deletion
mutations .(similar to figure 15-6) In the
modern era large-scale DRA sequencing .
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Topic four THE CODE IS
NEARLY UNIVERSAL
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1 . The functions of the universality of the
code
1 ) . It has had a huge impact in our
understanding of evolution as it made it possible
to directly compare protein coding sequences
among all organisms for which a genome sequence
is available . 2 ) . It also helped to
create the field of genetic engineering by making
it possible to express cloned copies of genes
encoding useful protein products in surrogate
host organisms .
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2 . The nature of the code is conservative ,
while the genetic code is in fact slightly
different from the standard code in certain
subcellular organelles .
1 ) . Sequences of the regions known to
specify proteins have revealed the following
differences between the standard and
mitochondrial genetic codes (table 15-6)
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?
UGA is not a stop signal but codes for
Trp . Internal methionine is encoded by
both AUG and AUA . In mammalian
mitochondria , AGA and AGG are not Arg codons
(of which there are six in the universal code )
but specify chain termination . In
fruit fly mitochondria , AGA and AGG ate also not
Arg codons but specify Ser .
?
?
?
?
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Table 15-6 Genetic Code of Mammalian Mitochondria
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2 ) . Mitochondrial tRNAs are likewise
unusual with respect to the rules by which they
decode mitochondrial messages . 3 ) .
Exceptions to the universal code are not
limited to mitrochondria but are also found in
several prokaryotic genomes and in the nuclear
genomes of certain eukaryotes .