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Initiation

Termination

Elongation

Chapter 14: Translation

Outline
Of all the events we have discussed,translation
is among the most highly conserved across all
organisms and among the most energetically
costly for the cell

Translation is a much more formidable challenge in


information transfer than the transcription of DNA into
RNA. Ulike the complementarity between the DNA
template and the ribonucleotides of the message RNA,
the side chains of amino acids have little or no specific
affinity for the purine and pyrimidine bases found in
RNA. Thus it seemed unlikely that direct interactions
between the mRNA template and the amino acids could
be responsible for the specific and accurate ordering of
amino acids in a polypeptide.

Four primary components:


1. mRNAs (~5% of total cellular RNA)
2. tRNAs (~15%)
3. aminoacyl-tRNA synthetases (tRNA
)
4. ribosomes (~100 proteins and 3-4 rRNAs-~80%)

Topic 1: mRNA
Polypeptide chains are specified by openreading frames
ORF:the protein coding region of each mRNA is
composed of a contiguous, non-overlapping
string of codons .
Each ORF specifies a single protein and starts
and ends at internal sites within the mRNA .
That is the ends of an ORF are distinct from the
ends of the mRNA.

The start codons


In becteria: usually AUG, sometimes
GUG ,even UUG
In eukaryotic cells: always AUG
Fuction :
1.Specifies the first amino acid to be
incorporated into the growing polypeptide
chain.

2.Defines the reading frame for all subsequent


codons.

The stop codons


Define the end of the open-reading frame and
signal termination of polypeptide synthesis.
Three kinds:
UAG
UGA
UAA

Two conceptions
Polycistronic mRNA: mRNAs containing multiple
ORFs
usually in prokaryotic cells
Monocistronic mRNA: mRNAs containing a
single ORF
usually in eukaryotic cells

RBS
Prokaryotic mRNAs have a ribosome binding
site that recruits the translational machinery.
RBS:a short sequence upstream of the start
codon in many prokaryotic open-reading frame.
Location:three to nine base pairs on the 5side
of the start codon.
Function:complementary to a sequence locate
near the 3 end of one of the RNA
components ,the 16S ribosomal RNA

One conception:
Translational coupling:
some prokaryotic ORFs internal to a polycistronic message
lack a strong ribosome binding site but are nonetheless
actively translated. In these cases the start codon often
overlaps the 3 end of the adjacent open-reading frame.
Thus ,a ribosome that has just completed translating the
upstream open-reading frame is appropriately positioned to
begin translating from the start codon for the downstream
open-reading frame,circumventing the need for a ribosome
binding site to recruit the ribosome.This phenomenon of linked
translation between overlapping open-reading frames is known
as Translational coupling

Eukaryotic mRNAs are modified at their 5and3


ends to facilitate translation
5cap: a methylated guanine nucleotide that is
joined to the 5end of the mRNA via an
unusual 5to 5linkage.
Function :once bound, the ribosome scans the
mRNA in a 5-3 direction to find the AUG
start codon.

Kozak sequence increases the translation efficiency.


Poly-A in the 3 end promotes the efficient recycling of ribosomes.

Topic 2: tRNA
.tRNAs are adaptors between codons and
amino acids
there are many types of tRNA molecules, but
each is attached to a specific amino acid and
each recognizes a particular codon, or codons.
75~95 ribonucleotides in length.

Features in common:
All tRNAs end at the 3 terminus with the
sequence 5-CCA-3 at the 3 end, where the
aminoacyl tRNA synthetase adds the amino
acid.
The presence of several unusual bases in their
primary structure: U, D and so on

Pseudouridine (U) is a modified base.


These modified bases in tRNA lead to
improved tRNA function

. tRNA share a common secondary structure


that resembles a cloverleaf
An acceptor stem
Three stem-loops
the U loop
the D loop
the anticodon loop
A variable loop

the secondary structure

tRNA secondary structure

Amino acid acceptor stem:

The 5-and 3-end


are largely basepaired to form the
amino acid
acceptor stem
which has no loop.

D-arm and D-loop

Composed of 3 or
4 bp stem and a
loop called the Dloop (DHU-loop)
usually
containing the
modified base
dihydrouracil.

Anticodon loop and


anticodon loop

Consisting of a 5 bp
stem and a 7
residues loop in
which the anticodon
is located. The 3-nt
anticodon sequence
is used to recognize
the codon sequence
in the mRNA

Variable arm and T-arm:


Variable arm:
3 to 21
residues and
may form a
stem of up to 7
bp.
T-arm
composed of a
5 bp stem
ending in a
loop containing
the invariant
residues GTC.

.tRNAs have an L-shaped three-dimensional


structrue

Three kinds of interaction stablize this Lshaped structure


Hydrogen bonds between bases in different
helical regions
Interactions between the bases and the sugarphosphate backbone
The additional base stacking gained from
formation of the two extended regions of base
pairing.

Topic 3: attachment of amino acids to


tRNA

Two steps:
Adenylylation: the amino acid reacts with ATP
to become adenylylated with the concomitant
release of pyrophosphate.
tRNA charging: the adenylylated amino
acid,which remains tightly bound to the
synthetase, reacts with tRNA.

There are two classes of tRNA


synthetases.
Class I: attach the amino acids to the 2OH
of the tRNA, and is usually monomeric.
Class II: attach the amino acids to the 3OH
of the tRNA, and is usually dimeric or
tetrameric

Adenylylation

tRNA charging

Two challenges of aminoacyl tRNA synthetases


Recognize the correct set of tRNAs for a
particular amino acid.
Charge all of these isoaccepting tRNAs with
the correct amino acid.
Both the processes must be carried out with high
fidelity

How to solve?
tRNA synthetases recognize unique structural
features of cognate tRNAs.
The acceptor stem
The anticodon loop

second genetic code

Aminoacyl-tRNA formation is very accurate


How?
Some amino acids differ substantially in size,
shape, and chemical groups.
To some similar amino acids like the Ile and
Val

Firstly ,Valyl tRNA synthetase can sterically


exclude isoleucine from its catalytic pocket
because isoleucine is larger than valine.
Secondly,some aminoacyl
tRNA synthetases use an
editing pocket to charge
tRNAs with high accuracy,
just like Ile and Val.

Topic 4: the ribosome

The ribosome is composed of a large and a


small subunit
The large subunit: containing the peptidyl
transferase center. Responsible for the
formation of peptide bonds.
The small subunit: containing the decoding
center. Charging tRNAs read or decode the
codon units of the mRNA.

The ribosome differs in prokaryotic


and eukaryotic cells

Ribosome cycles

In cells, the small and large ribosome subunits


associate with each other and the mRNA,
translate it, and then dissociate after each
round of translation. This sequence of
association and dissociation is called the
ribosome cycle.

Polysome/polyribosome
Although a ribosome can synthesize only one
polypeptide at a time, each mRNA can be
translated simultaneously by multiple
ribosomes.
So, an mRNA bearing multiple ribosomes

Peptide bonds are formed by transfer of the


growing peptide chain from peptidyl- tRNA to
aminoacyl-tRNA.

Two charged species of tRNAs :


The aminoacyl-tRNA: attached at its 3 end to
the carboxyl group of the amino acid.
The peptidyl-tRNA: attached in exactly the
same manner to the carboxyl-terminus of the
growing polypeptide chain.

Ribosomal RNAs are both structural and catalytic


determinants of the ribosomes
The ribosome has three binding site for tRNA.

A question!
Both the decoding center and the peptidyl
transferase center are buried within the intact
ribosome. Yet ,mRNA must be threaded
through the decoding center during translation,
and the nascent polypeptide chain must escape
from the peptidyl transferase center. How do
these polymers enter and exit the robosome?

! Channels through the ribosome allow the mRNA


and growing polypeptide to enter or exit the
ribosome.

Two narrow channels in the small subunit:


only wide enough for unpaired RNA to pass
though.
An exit path in the large subunit: for the newly
synthesized polypeptide chain to pass though,
the size of the channel limits the folding of the
growing polypeptide chain, only helix
allowed.

Topic 5: Initiation of translation


.the ribosome be recruited to the mRNA
Prokaryotic mRNA are initially recrutied to
the small subunit by base-paring to rRNA
Eukaryotic ribosomes are recruited to the
mRNA by the 5 cap.

.A charged tRNA be placed into the P


site of the ribosome.
A specialized tRNA charged with a modified
methionine binds directly to the prokaryotic
small subunit.

fMet-tRNAifMet

.the ribosome positioned over the start


codon
Three initiation factors direct the assembly of an
initiation complex that contains mRNA and the
initiator tRNA.
IF1.
IF2.
IF3.

IF1.preventing tRNAs from binding to the


portion of the small subunit that will become
part of the A site.
IF2.GTPase, interact with the three key
components of the initiation machinery: the
small subunit, IF1 and charged initiator tRNA.
IF3. binding to the small subunit and blocks it
from reassociating with a large subunit, or
from binding charged tRNAs.

Topic 6: Elongation of translation


.The correct aminoacyl-tRNA is loaded into
the A site of the ribosome.
Aminoacyl-tRNAs are delivered to the A site
by elongation factor EF-Tu.aminoacyl-tRNAs
do not bind to the ribosome on their own.
Instead they are escorted to the ribosome by
the elongation factor EF-Tu.

The ribosome uses multiple mechanisms


to select against incorrect aminoacyltRNAs.
Two adjacent adenine residues in the 16S
RNA component of the small subunit.(a)
The GTPase activity of EF-Tu.(b)
Proofreading that occurs after EF-Tu is
rreleased.(c)

.a peptide bond is formed between the


aminoacyl-tRNA in the A site and the
peptide chain that is attached to the
peptidyl-tRNA in the P site.
The ribosome is a ribozyme!

.the resulting peptidyl-tRNA in the


A site and its associated codon must be
translocated to the P site.
Peptide bond formation and the
elongation factor EF-G drive
translocation of the tRNAs and
the mRNAs.

Key points
EF-G drives translocation by displacing the
tRNA bonnd to the A site.
EF-Tu-GDP and EF-G-GDP must exchange
GDP for GTP prior to participating in a new
round of elongation.
A cycle of peptide bond formation consumes
two molecules of GTP and one molecule of
ATP.

Topic 6:termination of translation


Release factors terminate translation on
response to stop codons.
Two classes of release factors:
RF1.
RF2.
RF1. recognizes the stop codon UAG.
RF2. recognizes the stop codon UGA.
They can both recognizes the stop codon UAA.

Short regions of
class I release factors
recognize stop codons
and trigger release
of the peptidyl chain.

Topic 7:translation dependent regulation


of mRNA and protein stability
. In prokaryotes ,the SsrA RNA rescues
ribosomes that translate broken mRNAs
lacking a stop codon .
tmRNA: In prokaryotic cells, stalled ribosome
are rescued by the action of a chimeric RNA
molecular that is part tRNA and part
mRNA,called tmRNA.

SsrA:a 457-neclelotide tmRNA, including a


region at its 3end that strongly resembles
tRNAAla

. Eukaryotic cells degrade mRNAs that are


incomplete or have premature stop codons.
Nonsense mediated mRNA decay: When an
mRNA contains a premature stop codon
(nonsense codon), the mRNA is rapidly
degraded by nonsense mediated mRNA decay.
Pre-releasing the ribosome at the nonsense codon
prior to reaching the exon-junction complex
initiates a talk between the complex and
ribosome to remove the 5 cap from the
mRNA .

Nonstop mediated decay: rescuing ribosomes


that translate mRNAs lacking a stop codon.
The lack of a stop codon results in ribosome
translation into the poly-A tail to produce
poly-Lys at the C-terminus of the polypeptide;
the poly-Lys marks the newly synthesized for
rapid degradation.
The ribosome eventually stalls at the 3 end of
the mRNA, which is bound by the Ski7 protein
that triggers the ribosome dissociation and
recruits a 3-5 exonuclease activity to degrade
the nonstop mRNA.

Key points of the chapter

The main challenge of translation and the


solution.
The structure and function of the
translation machinery.
The process of translation.
translation dependent regulation of
mRNA and protein stability .