BI20M3THE GENETIC CODE
ANDTRANSLATION
LECTURE 1
AIMS: To review:
methods used to crack the code;
features of the code.
Lehninger: Chapter 27Instant Notes in Molecular Biology: Sections P, Q
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http://www.abdn.ac.uk/~bch118/index.htmTRANSLATION IS:
synthesis of polypeptide on a mRNA template,the linear sequence of amino-acids being determined by the linear sequence of 3-base codons,the latter being determined by the linear sequence of 3-base triplet code-words on a DNA template.
DNA RNA protein
DNA replication transcription translation
3 bases read in sequence on a DNA template: triplet.3 bases read in sequence on a mRNA template: codon.
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METHODS USED TO CRACK THE GENETIC CODE
(a) RNA with simple, defined sequences synthesised, e.g.
5’ ...AAAAAAAAAAAAAAAA… 3’
5’ ...ACACACACACACACAC… 3’
(b) these RNAs used as mRNAs in a system that synthesises polypeptide in the test-tube. (It contains ribosomes, tRNA, amino-acids, ATP and other ingredients seen later.)
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(c) either the polypeptides produced analysed,or the tRNAs that bind to the ribosomes analysed (this is easier to do).
(Particular tRNAs are assigned to particular amino-acids, and have to bind to the ribosome for the amino-acid to be incorporated into the polypeptide – see later).
Results:
Using the examples above,
5’ ...AAAAAAAAAAAAAAAA… 3’
as a mRNA, produced the polypeptide
… LysLysLysLysLysLys …
and only tRNA for lysine bound to ribosomes.
So the AAA codon must specify lysine.
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5’...ACACACACACACACAC… 3’
as a mRNA, produced the polypeptide
… ThrHisThrHisThrHis …
and only tRNAs for threonine and histidine bound to ribosomes.
So the only 2 possible codons, ACA and CAC, must specify threonine and histidine.
(This experiment by itself doesn’t allow you to say which specifies which).
Similar experiments, with other mRNAs of defined sequence, resolved the code-word assignments.
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FEATURES OF THE CODE
(a) Degeneracy.
Most amino-acids have >1 codon assigned to them.
20 coded amino-acids43 = 64 possible code-words61 specify particular amino-
acids3 (UAG, UGA, UAA, read 5’ to 3’) are
translation stop signals (see later).
(b) The code is non-overlapping and comma-less.
A sequence of bases is read
… ABC DEF GHI JKL … aa1 aa2 aa3 aa4
and not
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… ABC aa1
CDE aa2
EFG aa3 etc
or
… ABC D EFG H IJK … aa1 aa2 aa3
comma comma
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(c) The triplet nature of the code explains the effects of frame-shift mutations.
These arise from insertion or deletion of a piece of DNA.
Base-sequenceof ‘normal’ DNA
… ABC DEF GHI JKL MNO … aa1 aa2 aa3 aa4 aa5
A piece of DNA inserted
… ABC DEF XXX XXX XGH IJK … aa1 aa2 ? ? ? ?
From this point, succeeding code-words are out-of-frame (even after the inserted DNA), so the protein may well be defective.
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Note:
1 the 3-base nature of the code means that there is a 1 in 3 chance of information getting back in frame after the insertion;
2 a deletion, in this respect, produces the same effect as an insertion.
Much more common than frame-shifts are ‘point’ mutations, produced by single-base changes:
... ABC DEF GHX JKL MNO …
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E.g. in human haemoglobin chain position 6, normal protein has glutamate,
but sickle-cell haemoglobin has valine.
This is caused by a T to A change on the DNA
… CTC … (codes for glutamate)
… CAC … (codes for valine).
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(d) Degeneracy of the code minimises effects of mutations.
1 Why spread 61 triplets over 20 amino-acids? Why not have just 20 code-words, and 40-or-so triplets meaning nothing?
With a degenerate code, a point mutation is likely to change a triplet to another specifying an amino-acid, (so a protein may still be made), rather than to one meaning nothing, (in which case, no protein would be made).
2 Because of the non-random assignment of triplets to amino-acids (see L, p.1038; IN, p.261), it is quite possible that a point mutation will change a triplet to another assigned to the same amino-acid. This is a ‘silent’ (aka ‘synonymous’) mutation.
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Look at the genetic code-word dictionary (Lehninger, p.1038; Instant Notes in Molecular Biology, p.261) and pick out code-word assignments for individual amino-acids, to illustrate the point made in Section 2 above.
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3 Because the non-random assignment of triplets to amino-acids extends to groups of amino-acids of similar type, even if a point mutation changes a triplet to one meaning another amino-acid, it is likely to be similar to the originally-coded amino-acid.
This is a ‘conservative’ mutation.
The mutant protein may well function, possibly even better than the original.
Much evolution works at the molecular level in this way.
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Look at the genetic code-word dictionary (Lehninger, p.1038; Instant Notes in Molecular Biology, p.261) and pick out code-word assignments for sets of amino-acids with similar R groups, to illustrate the point made in Section 3 above.
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(e) The code is nearly universal.
The same assignment of code-words to amino-acids is used by all living organisms (and viruses).
There are minor exceptions:
in some bacteria, mitochondria,some unicellular eukaryotes.
Often the exceptions involve one of the 3 translation stop signals (particularly UGA) being used to specify an amino-acid.
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BI20M3THE GENETIC CODE
ANDTRANSLATION
LECTURE 2
AIMS: To define:‘open reading frame’.
To review:
tRNA as an adapter molecule;
aminoacyl tRNA synthetases.
Lehninger: Chapter 27
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Instant Notes in Molecular Biology: Sections P, QOPEN READING FRAMES
A sequence of bases has three possible reading frames:
… ABC DEF GHI JKL …
… . . A BCD EFG HIJ KL . …
… . AB CDE FGH IJK L
Which frame is used depends on recognition by the translation apparatus of a translation start signal. This is the sequence AUG. (We will see later how this acts to start the process.)
The bases are then read in groups of three until, in frame, a translation stop codon (UAA, UGA or UAG) is reached.
So, an ‘open reading frame’ on a mRNA looks like this:
5’ … XXX AUG XXX XXX XXX … XXX UAA XXX …3’
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or UAG or UGA
untranslated start translated stop untranslated
TRANSFER RNA (tRNA)
There is no direct, specific interaction between an amino-acid and the sequence of three bases encoding it.
This is not surprising: the two have very different structures.
For the genetic code to work, an adapter molecule is needed, to bring these different structures together
(rather like a 2-/3-pin electrical plug adapter).
The adapter is transfer (t) RNA.
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PROPERTIES OF tRNA
1 Acts as adapter between mRNA codon and amino-acid.
2 Small for a nucleic acid (73-93 nucleotides).
3 Like other RNA, is transcribed from DNA.
4 Comprises about 15% of total cell RNA.
5 Rich in unusual bases (made by modification of A,U,G,C).
6 Much intra-strand double-helicity.
7 All tRNAs have a similar 2-D structure ‘clover-leaf’and 3-D structure upper-case L
stabilised by intra-strand H-bonding.
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Look at Figures 27-12 and 27-13 in Lehninger, p.1050, or Figure 2 in Instant Notes in Molecular Biology, p.265, which show 2- and 3-dimensional structures of t-RNA.
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HOW tRNA ACTS AS AN ADAPTER
1 Activation of amino-acids.
NH2 adenine R - C - C - OH + P-P-P-ribose H O amino-acid ATP
PPi
NH2 O adenine
R – C – C – O – P – O – ribose
H O O-
aminoacyl AMP
(‘activated amino-acid’)
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2 Activated amino-acid is loaded onto 3’ end of a tRNA.
O
O P O -
O
H2C O Base
adenine
OH OH amino-acid – P - ribose
3’ end of a tRNA aminoacyl AMP
AMP
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O
O P O -
O
H2C O Base
O OH
C O
H2N – C – H
R
aminoacyl tRNA
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The same set of enzymes catalyses both steps, i.e.
1 activation;
2 loading.
They are aminoacyl tRNA synthetases
(i.e. they are named for step 2).
Each of the 20 coded amino-acids is assigned
at least one, specific aminoacyl tRNA synthetase
and
at least one, specific tRNA.
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Aminoacyl tRNA synthetases are very specific in their action.
Each catalyses activation of one, particular amino-acid, and then loads that amino-acid only onto the tRNA assigned to it.
This specificity is crucial, because one of the tRNA loops has a 3-base sequence, called the anticodon, that is complementary to the codon of the amino-acid assigned to that tRNA.
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We can now visualise the adapter role of tRNA between codon and amino-acid.
activated, loaded amino-acid
anticodon
codon on mRNA
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So, recognition of the mRNA codon is through its interaction, by specific base complementarity, with a tRNA anticodon
(tRNA acting as the ‘adapter’),
and NOT by interaction with the amino-acid itself, which, once loaded onto its tRNA, isn’t recognised by anything (i.e. it’s anonymous).
For this adapter system to work, the correct amino-acid MUST be loaded onto the correct tRNA.
So, the specificity of the aminoacyl tRNA synthetases is crucial.
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WOBBLE
With the adapter system just described,
61 tRNAs would be expected, each with a different anticodon, complementary to the 61 coding mRNA codons.
In fact, there are <61 tRNAs.
This is because several tRNAs recognise >1 codon.
This is possible because, in some anticodons, the base in position 3pairs with >1 codon base.
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3’ 5’
anticodon - - - X Y Z - - -
codon - - - X’ Y’ - - - 5’ 3’
specific base less specificcomplementarity pairing (‘wobble’)
Codon redundancy occurs mainly in this third position (Lecture 1), and all codons pairing with XYZ in the diagram above encode the same amino-acid.
What evolutionary advantage might ‘wobble’ confer?
It means that the codon-anticodon interaction, although strong and specific enough to allow the adapter function of tRNA,
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is not so strong that need for its disruption slows the process of translation.PROOF-READING BY AMINOACYL tRNA SYNTHETASES
Some of these enzymes ‘check’ that they have activated/loaded the correct amino-acid.
E.g. the E.coli enzyme for isoleucine sometimes activates and loads valine (it has a similar structure).
The enzyme has an additional site at which hydrolysis of the aminoacyl tRNA occurs, releasing free amino-acid. Valine fits this site better than isoleucine.
The overall fidelity of translation is about 1 mistake / 104 amino-acids polymerised.
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So, it’s less accurate than replication of DNA.
BI20M3THE GENETIC CODE
ANDTRANSLATION
LECTURE 3
AIMS: To review:
initiation of polypeptide synthesis;
formation of peptide bonds;
termination of polypeptide synthesis.
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Lehninger: Chapter 27Instant Notes in Molecular Biology: Sections P, Q
RIBOSOMES
These organelles are the site of codon-anticodon recognition, and of polypeptide synthesis.
Ribosomes have a generally similar structure in all cells.
In E. coli
30S subunit(16S RNA + 21 proteins)
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50S subunit(23S RNA,5S RNA +
36 proteins)S= Svedborg unit (to do with how fast particles sediment in an ultracentrifuge.)
DIRECTIONALITY IN TRANSLATION
DNA replication (recap)
DNA polymerised: 5’ 3’DNA template ‘read’: 3’ 5’
Transcription (recap)
RNA polymerised: 5’ 3’DNA template ‘read’: 3’ 5’
Translation
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Polypeptide polymerised N CmRNA template ‘read’ 5’ 3’
HOW AUG in a mRNA ACTS as a TRANSLATION START SIGNAL
AUG is (also) the codon for the amino-acid methionine (Met).
In E.coli,There are two tRNAs for Met.Both are loaded with activated Met.Both have an anticodon complementary to AUG.
In only one, the activated, loaded Met is converted to N-formyl Met (fMet)
C O C O
H2N CH OHC N CH
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(CH2)2 H (CH2)2
S S CH3 CH3
3’ end of tRNA, 3’ end of tRNA,loaded with Met loaded with fMet
It is this tRNA, loaded with fMet, that plays a crucial role at the start of polypeptide synthesis.
The other tRNA (loaded with Met) recognises AUG in the middle of a message, and inserts Met into the middle of a polypeptide.
What is it about the tRNA loaded with fMet that makes it active in polypeptide initiation?
It is the only tRNA that can bind directly to the peptidyl site of the 50S ribosome subunit (which we see later).
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THE START OF TRANSLATION
(a) mRNA binds to the 30S subunit.(At this stage, the two subunits are separate.)
The initial interaction is between 4-9 bases of the 16S RNA, and complementary bases, just to the 5’ side of an AUG codon.
It is these 4-9 bases that differentiate the AUG from other AUGs within the message.
(b) tRNA, loaded with fMet, binds to the 30S subunit/mRNA complex,interacting, through its anticodon, with the AUG.
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Processes (a) and (b) need three proteins (IF-1, IF-2, IF-3) and GTP.
(c) The 50S subunit binds to the 30S subunit.
The fMet – loaded tRNA is now on the peptidyl site of the 50S subunit.
Process (c) needs GTP dephosphorylation.
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Look at Lehninger (pp.1056-1057; Figure 27-20) or Instant Notes in Molecular Biology (p.275; Figure 1), which give details of the three initiation steps of polypeptide synthesis outlined above.
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FORMATION OF THE PEPTIDE BOND
The three, repeated steps occur:
Step 1
tRNA, loaded with amino-acid, binds to the aminoacyl site.
This requires 2 proteins (EF-Tu, EF-Ts) andGTP dephosphorylation.
Step 2
The peptide bond is formed.
This requires ‘peptidyl transferase’, a ribozyme activity of the 23S RNA.
Step 3
The ribosome moves along the mRNA
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in a 5’ to 3’ direction.
This requires a protein (EF-G)and GTP dephosphorylation.Formation of the peptide bond.
(Lehninger, pp.1058-1061; Figures 27-23, 27-24, 27-25;Instant Notes in Molecular Biology, p.276; Figure 2).
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Notice that all the loaded tRNAs bind first to the aminoacyl site, and then move across to the peptidyl site.
To start the process, ONE tRNA must bind directly to the peptidyl site.
The only one that can do this is the tRNA loaded with fMet (as we saw earlier).
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TRANSLATION TERMINATION
When UAA, UAG or UGA occur, in frame, in a message,
one or other of three proteins (RF1, RF2, RF3) bind to the ribosome.
This causes hydrolysis of the bond between the last amino-acid of the polypeptide and the last tRNA.
Free polypeptide and free tRNA are released,
and the 30S and 50S subunits dissociate.
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