Download ribosome binding site Prokaryotic mRNAs have a ribosome binding

Ch14 Translation
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Messenger RNA
Transfer RNA
Attachment of amino acids to tRNA
The ribosome
Initiation of translation
Translation elongation
Termination of translation
Regulation of translation
Translation-dependant regulation of mRNA and
protein stability
Messenger RNA
Polypeptide chains are specified by open-reading frames
Fig 14-1 Three possible reading frames of the E. coli trp leader sequence
• 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.
Fig 14-2 Structure of mRNA
RBS: ribosome binding site
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.
yU
Fig 14-3 A subset of modified nucleosides found in tRNA
D
tRNAs share a common
secondary structure
that resembles a
cloverleaf
Fig 14-4 cloverleaf
representation of the 2nd
structure of tRNA
(1) The acceptor stem
(2) yU loop: 5’-TyUCG-3’
(3) D loop
(4) anticodon loop
(5) variable loop: 3-21 bases
tRNAs have an L-shaped 3-D structure
Fig 14-5 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
Fig 14-6
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
Fig 14-7
Fig 14-8 co-crystal structure of
glutaminyl aminoacyl tRNA
synthetase with tRNAgln
Aminoacyl-tRNA formation is very accurate
Fig 14-9
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
Fig 14-10 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.
Fig 14-11 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
Fig 14-12 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.
Fig 14-13 Composition of
the prokaryotic and
eukaryotic ribosomes.
The large and small subunits undergo association and
dissociation during each cycle of translation
Fig 14-14 Overview of the events of translation
Fig 14-15 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
Fig 14-16 The peptidyl transferase
reaction
The ribosome catalyzes a single
chemical reaction:
The formation of a peptide bond
Fig 14-17 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
Fig 14-18
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 (Fig 14-19)
Fig 14-20 The interaction
between the A site and P site
tRNAs and the mRNA within
the ribosome.
Fig 14-21 The polypeptide
exit center
The initiation of translation
Fig 14-22 An overview of the
events of translation initiation
Prokaryotic mRNAs are initially recruited to the
small subunit by base-pairing to rRNA
Fig 14-23 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
Fig 14-24 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.
Fig 14-25 A summary of
translation initiation in
prokaryotes
Eukaryotic ribosomes are recruited to the mRNA by the 5’ Cap
Fig 14-27 assembly of the eukaryotic
small ribosomal subunit and initiator
tRNA onto the mRNA
eIF4B: helicase; unwinding any RNA
secondary structure
Fig 14-27 identification of the initiating
AUG by the eukaryotic small ribosomal
subunits
The start codon is found by
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
Fig 14-28 a model for the
circularization of eukaryotic
mRNA, through the
interaction between eIF4G
and poly-A binding protein.
Translation elongation
Fig 14-29 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.
Fig 14-30 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?
Fig 14-35
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
Fig14-6
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 peptidyltransferase center
SPF: peptide anticodon; for
interacting with stop codon
GDP-GTP exchange and
GTP hydrolysis control the
function of the class II
release factor
Fig 14-39 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
Fig 14-40 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.
Fig 14-39 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
Fig 14-40
Exosome: 3’-5’ exonuclease