Translation

Messenger RNA
Polypeptide chains are specified by open-reading frames
• Open reading frame (ORF):
a contiguous string of codons that specify a single
protein; read in a particular frame (as set by the first
codon) that is open to translation. ORF starts and ends
at internal sites within the mRNA.

• Start codon in eukaryotes: AUG

• Stop codons: UAG, UGA, UAA

• Eukaryotic mRNAs almost always contain a single

ORF, whereas prokaryotic mRNAs contain one or
more ORF.
• Polycistronic mRNA: mRNA that contain multiple
ORF.

• Monocistronic mRNA: mRNA that contain single ORF.

Prokaryotic mRNAs have a ribosome binding site that
recruits the translational machinery

• Ribosome binding site = Shine-Dalgarno sequence

1. 3-9 bp on the 5’ side of the start codon

2. Complementary to a sequence near the 3’ end of 16s

rRNA.
Eukaryotic mRNAs are modified at their 5’ and 3’ ends to
facilitate translation
• 5’ modifications:

(1) Eukaryotic mRNAs recruit ribosomes using 5’ cap.

5’ cap: methylated G nucleotide that is linked to 5’
end of mRNA by 5’-5’ linkage

• 5’ cap recruits ribosome to the mRNA; the ribosome

bound to mRNA moves in a 5’ to 3’ direction until it
encounters a start codon (scanning).

(2) Kozak sequence (5’-G/ANNAUGG-3’): thought to

interact to with initiator tRNA
• 3’ modifications:

Poly-A tail enzymatically added by poly-A

polymerase.

enhance translation by promoting efficient

recycling of ribosomes.
Transfer RNA
tRNAs are adaptors between codons and amino acids

• tRNA: between 75 to 95 ribonucleotides

• tRNA end at 3’-terminus with the sequence CCA,

where the cognate amino acid is attached.

• Unusual bases are present in tRNA structure.

 U D

A subset of modified nucleosides found in tRNA

 tRNAs share a common
secondary structure that
resembles a cloverleaf

cloverleaf representation of the

2nd structure of tRNA

(1) The acceptor stem

U loop: 5’-TUCG-3’
(3) D loop
(4) anticodon loop
(5) variable loop: 3-21 bases
tRNAs have an L-shaped 3-D structure

Conversion between the cloverleaf and the actual 3-D structure of a tRNA
Attachment of amino acids to tRNA
tRNAs are charged by the attachment of an amino acid to
the 3’ terminal adenosine nucleotide via a high-energy
acyl linkage

• Charged tRNA

• Uncharged tRNA
Aminoacyl tRNA synthetase charge tRNAs in two steps

Adenylylation of amino acid

Transfer of adenylylated amino acid to tRNA: tRNA charging
• Each aminoacyl tRNA
synthetase attaches a
single amino acid to one or
more tRNAs
isoaccepting tRNA

• tRNA synthetase recognize

unique structural features
of cognate tRNAs
Co-crystal structure of
glutaminyl aminoacyl tRNA
synthetase with tRNAgln
Aminoacyl-tRNA formation is very accurate

Some aminoacyl tRNA

synthetases use an editing (as
a molecular sieve) pocket to
charge tRNAs with high
fidelity
The ribosome is unable to discriminate between
correctly and incorrectly charged tRNAs

cysteinyl-tRNA charged with

C or A
The Ribosome
• Rate of DNA replication: 200-1000 nt/sec

• Rate of translation in prokaryotes: 2-20 amino acids/sec

• Rate of translation in eukaryotes: 2-4 amino acids/sec

In prokaryotes, the transcription and translation
machineries are located in the same compartment.

Prokaryotic RNA
polymerase and the
ribosome at work on the
same RNA
In eukaryotes, transcription happens in the nucleus while
translation happens in the cytoplasm.
The ribosome is composed of a large and a small subunit

Sedimentation by ultracentrifugation to separate individual ribosome

subunits and the full ribosomes.
S: Svedberg (sedimentation velocity) determined by both size and shape.
• Large subunit contains peptidyl transferase center (for
formation of peptide bond)

• Small subunit contains decoding center.

Composition of the
prokaryotic and eukaryotic
ribosomes.
The large and small subunits undergo association and
dissociation during each cycle of translation

Overview of the events of translation

An mRNA bearing multiple ribosomes is known as a polyribosome
or a polysome.
New amino acids are attached to the C-terminus of the
growing polypeptide chain

Peptides bonds are formed by transfer of the growing

polypeptide chain from one tRNA to another
The peptidyl transferase reaction

The ribosome catalyzes a single

chemical reaction:

The formation of a peptide bond

two views of the ribosomes

• Ribosomal RNAs are both structural and catalytic

determinants of the ribosome.
• Most ribosomal proteins are on the periphery of the
ribosome, while the functional core of ribosome is composed
mostly from rRNA.
The ribosome had three binding sites for tRNA

A: for aminoacylated-tRNA
P: for peptidyl-tRNA
E: for exit
Channels through the
ribosome allow the mRNA
and growing polypeptide to
enter and/or exit the
ribosome

The interaction between the A

site and P site tRNAs and the
mRNA within the ribosome.
The polypeptide exit center
The initiation of translation

An overview of the events of

translation initiation
Prokaryotic mRNAs are initially recruited to the
small subunit by base-pairing to rRNA

The 16S rRNA interacts

with the RBS to position
the AUG in the P site.
 A specialized tRNA charged with a modified methionine
binds directly to the prokaryotic small subunit

• Initiator tRNA: fMet-tRNAifMet (base-pairs with AUG or GUG)

• Deformylase removes the formal group during or after the
synthesis

methionine and N-formyl methionine

Three initiation factors direct the assembly of an initiation
complex that contains mRNAs and the initiator tRNA
A model of initiation factor
binding to the 30S ribosomal
subunit.

IF1: prevents tRNA from

binding to A site

IF2: a GTPase; interacts with

initiator tRNA and IF1, and
thus prevents further tRNA
binding to small subunits.

IF3: binds to small subunit

and prevent it from
reassociating with large
subunit; essential for
translation initiation.
A summary of translation
initiation in prokaryotes
Eukaryotic ribosomes are recruited to the mRNA by the 5’ Cap

assembly of the eukaryotic small

ribosomal subunit and initiator tRNA
onto the mRNA

eIF4B: helicase; unwinding any RNA

secondary structure
identification of the initiating AUG by The start codon is found by
the eukaryotic small ribosomal subunits scanning downstream from the 5’
end of the mRNA
uORF: short, upstream, open-reading frame, less than 10 amino acids long
IRES (internal ribosome entry
site)
Translation initiation factors hold eukaryotic mRNAs in circles

a model for the circularization

of eukaryotic mRNA, through
the interaction between eIF4G
and poly-A binding protein.
Translation elongation

summary of the steps of

translation

The mechanism of elongation

is highly conserved between
prokaryotes and eukaryotes.
Aminoacyl-tRNA are
delivered to the A site by
elongation factor EF-Tu.

EF-Tu escorts aminoacyl-tRNA

to the A site of the ribosome.

The interaction between EF-Tu

and factor binding site of large
subunit triggers the GTPase of
EF-Tu.
The ribosome uses multiple mechanisms to select against incorrect
aminoacyl-tRNAs.
Minor groove interactions
The ribosome is a ribozyme: peptidyl transferase reaction is
catalyzed by RNA, mainly 23S rRNA.
Peptide bond formation and the elongation factor EF-G
drive translocation of the tRNAs and the mRNA
Hybrid state of tRNA exposes factor-binding site;
EF-G can only bind to ribosome by a GTP-bound
form.

EF-G drives translocation by

displacing the tRNA bound to
the A site
How does EF-G-GDP interact with the A site so effectively?

Left: EF-Tu-GDPNP-Phe-tRNA
Right: EF-G-GDP
EF-Tu-GDP and EF-G-GDP must exchange GDP for GTP
prior to participating in a new round of elongation

GDP has a lower affinity for EF-G than GTP

For EF-Tu, a GTP-exchange factor EF-Ts is required for

the GDP-GTP exchange.

A cycle of peptide bond formation consumes two molecules

of GTP and one molecule of ATP
Termination of translation
release factors terminate translation in response
to stop codons

Release factors (RF) activates the hydrolysis of polypeptide from the

peptidyl-tRNA

Class I RF: recognizes stop codon

Class II RF: stimulate dissociation of class I RF from ribosome

Class I RF:
prokaryotes: RF1 (UAG, UAA); RF2 (UGA, UAA)
eukaryotes: eRF1 (UAG; UGA; UAA)
Class II RF: regulated by GTP
prokaryotes: RF3
eukaryotes: eRF3
Short regions of class I release factors recognize stop codons
and trigger release of the peptidyl chain

Model of a RF1 bound to the A

site

GGQ: involved in polypeptide

hydrolysis; close to peptidyl-
transferase center

SPF: peptide anticodon; for

interacting with stop codon
GDP-GTP exchange and
GTP hydrolysis control the
function of the class II
release factor

polypeptide release is mediated

by two RF

RF-3 has a higher affinity to

GDP than to GTP
The ribosome recycling
factor (RRF) mimics a tRNA

RRF and EF-G combine to

stimulate the release of tRNA
and mRNA from a terminated
ribosome.

RRF is only associated with the

large subunit of the A site.
Puromycin terminates translation by mimicing a tRNA in the A site.
Regulation of translation
• Although the expression of most genes is regulated at the level of
mRNA transcription, it is more effective for the cell to regulate gene
expression at the level of translation.

• As with other types of regulation, translational control typically

functions at the level of initiation.
Protein or RNA binding near the ribosome-binding site
negatively regulates bacterial translation initiation
Regulation of prokayryotic translation: ribosomal proteins
are translational repressors of their own synthesis
Global regulators of eukaryotic translation target key
factors required for mRNA recognition and initiator tRNA
ribosome binding
Spatial control of translation by mRNA-specific 4E-BPs
An iron-regulated, RNA-binding protein controls
translation of ferritin
Translation of the yeast transcriptional activator Gcn4 is controlled
by short upstram ORFs and ternary complex abundance

Fig 14-48
Translation-dependent regulation of mRNA and protein
stability

Being single-stranded, mRNAs are more susceptible to breakage.

Such damaged mRNAs have the possibility of making incomplete
or incorrect proteins.
 The SsrA RNA rescues ribosomes that translate broken
mRNAs

• tmRNA: in prokaryotic cell, stalled ribosomes are

rescued by a chimeric RNA (part tRNA and part
mRNA)

• SsrA is a 457 nt tmRNA that includes a 3’ end strongly

resembles tRNAala.
The tmRNA and SsrA rescue
ribosomes stalled on prematurely
terminated mRNAs.

How does the SsrA RNA bind to

only stalled ribosomes??

Large size
Eukaryotic cells degrade mRNAs that are incomplete or
that have premature stop codon
Exosome: 3’-5’ exonuclease