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Translation

Vipin Shankar
The Central Dogma
Translation : overview

Genetic information within the order of the
nucleotides in mRNA is used to generate the linear
sequences of amino acids in protein.
Overview…
 Most highly conserved event across all
organisms.
 One of the most energetically costly event for the
cell.
 More than 80% of the cell’s energy and 50% of
dry weight is dedicated to protein synthesis in a
rapidly growing cell.
 Synthesis of a single protein requires the
coordinated action of over a 100 proteins and
RNAs.
Challenge
 Translation is a more formidable challenge than
transcription.
 Side chains of amino acids have little or no
specific affinity to the nucleotide bases of mRNA.
 Direct interaction between mRNA template and
amino acids could not be responsible for accurate
ordering of amino acids in protein.
Adaptor molecule
 In 1955, Francis H Crick, proposed that
prior to their incorporation into
polypeptides, amino acids must attach to a
special molecule that is capable of directly
interacting with and recognizing the three-
nucleotide-long coding units of the mRNA.
 2 years later Paul C Zamecnik and Mahlon
B Hoagland discovered tRNA.
The Machinery
 mRNA.
 tRNA.
 Aminoacyl tRNA synthetase.
 Ribosome.
 Factors
Messenger RNA
 The translation machinery decodes only a portion
of the mRNA.
 Information for protein synthesis is in the form of
nucleotide triplets – Codons.
 The coding region is composed of contiguous,
non-overlapping string of codons called Open
Reading Frame (ORF).
 Each ORF specifies a single protein and starts and
ends at internal sites within the mRNA.
Messenger RNA…
 Translation starts at the 5’ end of the ORF and
proceeds one codon at a time to the 3’ end.
 The first and the last codons of an ORF are called
the start & stop codons respectively.
 The start codon has 2 important functions:
 Specifies the amino acid to be incorporated into the
growing polypeptide.
 Defines the reading frame for all subsequent codons.
Polycistronic & Monocistronic mRNA
 mRNAs contain at least one ORF.
 The number of ORFs vary in prokaryotes &
eukaryotes.
 Prokaryotic mRNA usually contain 2 or
more ORFs & hence encodes for multiple
polypeptide chains – polycistronic mRNA.
 Eukaryotic mRNA generally have a single
ORF – monocistronic mRNA.
The Ribosome Binding Site (RBS)
 For translation to occur, the ribosome must be recruited to
the mRNA.
 Many pk. ORFs contain a short sequence typically located
3 -9 bps upstream of the start codon – the RBS, or the
Shine- Dalgarno sequence.
 The RBS is complimentary to a sequence located near the
3’ end of one of the RNA components of the ribosome
(16s rRNA).
 The core sequence is 5’ – CCUCCU – 3’
 The extent of complementarity and the spacing between
the RBS & the start codon influence the rate of translation.
Translational coupling
 In some pk. ORFs internal to a polycistronic
message lack a strong RBS, but are nonetheless
actively translated.
 In these cases the start codon often overlaps the 3’
end of the adjacent ORF (5’ –AUGA- 3’)
 A ribosome that has just completed translation of
the upstream ORF is appropriately positioned to
begin translating from the start codon of the
downstream ORF, circumventing the need for
fresh ribosome recruitment.
Ek. mRNAs are modified
 Ek. mRNAs recruit ribosomes using a chemical
modification called the 5’-cap.
 The 5’ cap is a methylated guanine nucleotide that
is joined to the 5’ end of the mRNA through a
5’-5’ linkage.
 The resulting structure recruits the ribosome to the
mRNA.
 Once bound the ribosome moves in a 5’-3’
direction until it encounters a start codon –
Scanning.
Ek. mRNA…
 In some mRNAs, a purine base is present 3
bases upstream of the start codon and a ‘G’
immediately downstream.
(5’-G/ANNAUGG- 3’) – Kozak sequence.
 Its presence increases the efficiency of
translation.
 This sequence interact with initiator tRNA.
Ek. mRNA…
 At the extreme 3’ end of the mRNA a chain
of Adenine nts are present – the poly A tail.
 This tail is added enzymatically by the
enzyme poly-A polymerase.
 Despite its location at the 3’ end, the poly-A
tail enhances the rate of translation, by
promoting efficient recycling of ribosomes.
Transfer RNA
 tRNAs are adaptors between codons and
amino acids.
 There are many types of tRNAs, but each is
attached to a specific amino acid and each
recognizes a particular codon, or codons.
 All though the exact sequence varies, all
tRNAs have certain features in common.
Common features of tRNAs
 All tRNAs end at 3’ end with the sequence 5’ –
CCA- 3’
 This is the site that is attached to the cognate
amino acid.
 Several unusual bases are present in the tRNAs’
primary structure.
 The unusual bases are created post-
transcriptionally, by enzymatic modifications of
the normal bases.
Attachment of amino acid to tRNA
 Charging of tRNA.
 Acyl linkage between, the carboxyl group of the
amino acid and the 2’ or 3’ hydroxyl group of the
adenosine nucleotide that protrudes from the
acceptor stem.
 This is high-energy bond, and the hydrolysis of
this bond results in large change of free energy.
 The energy released when the bond is broken
drive the formation of peptide bond during
translation.
Charging…
 Aminoacyl tRNA synthetase, attach an
amino acid to a tRNA in a 2 step reaction.
 Step 1: Adenylylation: amino acid reacts with
ATP to become adenylylated with the
concomitant release of pyrophosphate.
 Adenylylation refers to the transfer of AMP,
opposed to adenylation which indicates the
transfer of adenine.
Charging…

PPi
Charging…
 As a result of adenylylation, the amino acid
is attached to adenylic acid via a high-
energy ester bond in which the carbonyl
group of the amino acid is joined to the
phosphoryl group of AMP.
 The adenylylated amino acid remain tightly
bound to the synthetase.
Charging…
 Step 2: adenylylated amino acid is
transferred from the enzyme to the 3’ end of
the tRNA via the 2’- or 3’ – hydroxyl and
the concomitant release of AMP.
 There are 2 classes of tRNA synthetases.
 Class I enzymes attach amino acids to 2’ OH of
tRNA and are generally monomeric.
 Class II enzymes attach amino acids to 3’ OH
of tRNA and are typically dimeric or trimeric.
General Structure of amino acyl tRNA
Charging…
 Each of the 20 amino acids is attached to the
appropriate tRNA by a single, dedicated tRNA
synthetase.
 Because, most amino acids are specified by more
than one codon, it is not uncommon for one
synthetase to recognize and charge more than one
tRNA: Isoaccepting tRNAs.
 Nevertheless, the same tRNA synthetase is
responsible for charging all tRNAs of a particular
amino acid.
Specificity of charging
 tRNA synthetases:
 Must recognize the correct set of tRNAs for a
particular amino acid, (specificity of tRNA
recognition) and
 must charge all these isoaccepting tRNAs with
the same amino acid (Accuracy of amino acyl
tRNA formation).
Specificity of tRNA recognition
 tRNA synthetases recognize some specific
regions of the tRNA which help them to
identify the correct cognate tRNA –
specificity determinants.
 Specificity determinants are clustered at 2
distinct sites on the tRNA-
 the acceptor stem - discriminator.
 the anti-codon loop – anti-codon.
Specificity of tRNA recognition…
 The anti-codon dictates the amino acid, that the
tRNA is responsible for incorporating.
 However, because each amino acid is usually
specified by more than one codon, recognition of
anti-codon cannot be used in all cases.
 So the discriminator plays a greater role in tRNA
recognition.
 The set of tRNA determinants that enable
synthetases to discriminate among tRNAs is
referred to as ‘the second genetic code’.
Accuracy of amino acyl tRNA formation
 The challenge to recognize the correct
amino acid is even more daunting.
 The relatively small sizes and the similarity
makes the task difficult.
 Despite this challenge, the frequency of
mischarging is very low: typically less than
1 in 1000 tRNAs is charged with incorrect
amino acid.
Accuracy of amino acyl tRNA formation...
 Different synthetases use different
mechanisms to distinguish between the
amino acids.
 Eg 1.
 Tyrosyl tRNA synthetase: the oppertunity of
forming a strong and energitically favourable
hydrogen bond with the hydroxyl moiety of
tyrosine helps the synthetase to identify
tyrosine.
Accuracy of amino acyl tRNA formation...
 Eg 2.
 Isoleucine and valine differ only by a single methyl
group.
 Valyl tRNA synthetase can sterically exclude
isoleucine from its catalytic pocket as isoleucine is
larger than valine.
 Eg 3.
 But valine can easily slip into the isolucyl tRNA
synthetase catalytic pocket.
 The interaction of the methyl group on isoleucine gives
an extra -2 to -3 Kcal/mol of free energy. This
relatively small energy difference makes the reaction
100 times more likely.
Accuracy of amino acyl tRNA formation...
 One common mechanism to increase fidility of an
amino acyl tRNA synthetase is to proofread the
products of the charging reaction.
 This is performed by an editing pocket present in
addition to the catalytic product.
 All wrongly charged amino acyl tRNAs enter the
editing pocket, while the correctly charged amino
acyl tRNA is sterically excluded (molecular
sieve).
 Within the editing pocket the amino acyl tRNA is
hydrolyzed.
Accuracy of amino acyl tRNA formation...

 The ribosome is unable to discriminate
between the correctly and incorrectly
charged tRNAs.
 The ribosome ‘blindly’ accepts any tRNA
that exhibits a proper codon – anti codon
interaction, whether or not the tRNA carries
its cognate amino acid.
The Ribosome
 A macromolecular machine that directs the
synthesis of proteins.
 Ribosome is larger and more complex than the
minimal machinery required for DNA and RNA
synthesis.
 Ribosome is composed of at least 3 different
RNAs and 50 different proteins with an overall
molecular mass of over 2.5 mega daltons.
 The rate of translation is very slow (2-20 aa/sec).
Ribosome…
 Composed of a large and a small subunit.
 Large subunit contains the peptidyl transfer center,
responsible for the formation of the peptide bond.
 The small subunit contains the decoding center, in
which the charged tRNAs read or ‘decode’ the
codon units of the mRNA.
 The large and small subunits are named according
to the velocity of their sedimentation when
subjected to a centrifugal force (Svedberg unit).
The ribosome cycle
 Central to the mechanism of translation is a
cycle in which the small and the large
subunits of the ribosome associate with
each other and the mRNA, translate the
target mRNA, then dissociate after each
round of protein synthesis: ribosome cycle.
The polysome
 Although a ribosome can synthesize only
one polypeptide at a time, each mRNA can
be translated simultaneously by multiple
ribosomes.
 An mRNA containing multiple ribosomes is
called polyribosome or polysome.
tRNA binding sites
 Ribosome has 3 tRNA binding sites called
A, P and E sites.
 A site for amino acyl tRNA.
 P site for peptedyl tRNA.
 E site is the exit site.
The process of translation
 3 steps
 Initiation.
 Elongation.
 Termination.
Initiation of translation
 3 events
 Recruitment of ribosome.
 Placement of charged tRNA into the P site of the
ribosome.
 Precise positioning of the ribosome over the start
codon.
 The positioning of the ribosome over the start
codon is critical since it establishes the reading
frame for the translation of mRNA.
Initiator tRNA.
 First charged tRNA to enter the ribosome.
 Base pairs with the start codon AUG or
GUG.
 AUG and GUG have different meanings
within the ORF (met & val).
 Initiator tRNA is charged with N-formyl
methionin (fmet).
 Initiator tRNA depicted as fMet-tRNAifMet
Initiation factors
 IF1, IF2 & IF3
 IF1- prevents initiator tRNA from binding to A
site.
 IF2 – a GTPase interacts with the small subunit,
IF1 and initiator tRNA. Facilitates subsequent
association of initiator tRNA with the small
subunit and prevents other charged tRNAs from
binding.
 IF3 – binds with small subunit and prevents its
from associating with the large subunit.
Initiation in prokaryotes
 Step 1: IF3 binds with small subunit of
ribosome near the E site.
 Step 2: IF1binds the small subunit of
ribosome near the A site.
 Step 3: IF2 in association with a GTP
molecule bids to IF1.
 Step 4: The mRNA and the fMet-tRNAifMet
associate with the assembly.
Initiation in prokaryotes…
 Step 5: Ribosome positioning by
complimentary pairing of 16s rRNA and the
Shine Dalgarno Sequence.
 This complex is called the 30s initiation
complex.
 Step 6: IF3 falls off. Large subunit
associates with small subunit.
Initiation in prokaryotes…
 Step 7: hydrolysis of GTP by IF2 is
initiated by the factor binding center of the
large subunit.
 IF2 – GDP has less affinity to ribosome
hence IF2, GDP and IF1 are released.
 This is the 70s initiation complex.
mRNA

70s initiation complex 30s initiation complex
Initiation in eukaryotes
 Similar to that in prokaryotes.
 More number of initiation factors present.
 A little more complex than in prokaryotes.
 Initiator tRNA is charged with methionine
(met).
Initiation in eukaryotes…
 Step 1: factors eIF6, eIF3 & eIF1A (analogous to IF3 &
IF1 in pk), bind to ribosome- ribosome dissociates into
small and large subunits.
 Step 2 : eIF2 – GTP forms complex with initiator tRNA
[eIF2-GTP- Met-tRNAiMet].
 Step 3: eIF5B – GTP associates with small subunit of
ribosome in a eIF1A dependent manner.
 Step 4: eIF5B helps to recruit [eIF2-GTP- Met-tRNAiMet]
complex to the small subunit.
 The initiator tRNA is positioned in the P site of the small
subunit.
 This complex is called the 43S-pre-initiation complex.
Initiation in eukaryotes…
 Step 5: eIF4E subunit of eIF4 binds to the 5’ cap
of mRNA. During the same step, 2 other subunits
of eIF4 binds with the mRNA non-specifically –
forming mRNA-eIF4 complex.
 Step 6: eIF4B activates helicase activity in another
subunit of eIF4.
 The helicase unwinds any secondary structures that
may have formed in the mRNA.
 Removal of secondary structures is critical for the
binding of the small subunit of the ribosome.
Initiation in eukaryotes…
 Step 7: The mRNA – eIF4 complex
associates with the 43s pre-initiation
complex through the interaction between
eIF4F and eIF3.
 This is now called the initiation complex.
Initiation in eukaryotes…
 Step 8: The initiation complex move along the
mRNA in a 5’-3’ direction in an ATP dependent
process, driven by the eIF4 associated RNA
helicase.
 During this movement, the small subunit scans the
mRNA for the first start codon.
 The start codon is recognized by the base pairing
between the anti codon of the initiator tRNA and the
start codon.
 Kozak sequence points out the correct start codon.
Initiation in eukaryotes…
 Step 9: Correct base pairing triggers the release of
eIF2 and eIF3.
 Step 10: The large subunit attaches to the small
subunit.
 Step 11: The remaining factors are released by
stimulating GTP hydrolysis by eIF5B.
 Step 12: The initiator tRNA is placed in the P site.
 This is the 80s initiation complex.
Exceptions to normal initiation
 uORFs
 Not all ek polypeptides are encoded by an ORF that starts with the
AUG that is the most proximal to the 5’ terminus.
 In some cases, the first AUG is not a proper sequence context,
resulting in its bypass.
 In other cases, short, upstream ORF (uORF) encoding peptides
less than 10 aa, are found upstream to the principal ORF.
 The uORF act to regulate the rate of translation of the downstream
ORF.
 The uORF is followed by a sequence of RNA that retains the
initiation complex, which continue scanning of the mRNA for
AUG
Exceptions to normal initiation
 IRES
 Internal Ribosome Entry Sites (IRES) are RNA
sequences that function like the pk ribosome
binding sites.
 They recruit the small subunit to bind and
initiate at an internal site in the mRNA.
 This method is also exhibited by viral mRNAs.
Translation initiation factors hold ek
mRNA in circles.
 In addition to binding the 5’ end of ek mRNA, the
initiation factors are closely associated with the 3’
end of the mRNA through its poly-A tail.
 This is mediated by an interaction between eIF4F
and Poly-A binding protein that coats the poly-A
tail.
 These interactions hold the mRNA in a circular
configuration via a protein bridge between the 5’
and 3’ ends of the molecule.