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Molecular Machines
Autumn 2013 Michael Pavlov
Based on chapter 8.4, MCB book
Lecture 3: Mechanisms of Translation.
•General features of the ribosome: special roles played by 30S and 50S subunits
•tRNA and tRNA synthetases
•The elongation cycle of the ribosome: accuracy of translation.
•Termination of protein synthesis.
•Initiation of protein synthesis in prokaryotes.
•Basic initiation factors and regulation of translation initiation in eukaryotes.
•Regulation of the formation of 43S complex by eIF2 phosphorylation.
•mRNA recruitment to the 40S subunit: the role of the 5’-cap structure and eIF4F.
•Translation regulation at mRNA level
•Initiation on internal ribosome binding sites: IRES
•miRNAs in post-transcriptional mRNA regulation
•mRNAs that code for non-functional proteins are eliminated by NMD
General features of the ribosome: Roles played by 30S and 50S subunits.
Figure 4-26. A low-resolution structure of
Ecoli 70S ribosome.
Prokaryotic and eukaryotic ribosomes are
very similar but the eukaryotic ribosome is
larger and has some additional features.
The ribosome has two subunits with
different functions.
•The ribosome consists of two subunits that
separate after the termination of mRNA
translation and then re-assemble again during
the initiation of translation.
•The small ribosomal subunit in prokaryotes
consists of 16S RNA and more than 20
proteins. It has a sedimentation coefficient
30S and that is why it is called the 30S
subunit.
•The large 50S subunit in prokaryotes
consists of 23S rRNA+5S rRNA and more
than 30 proteins.
•The assembled ribosome is called the 70S
ribosome in bacteria.
•In eukaryotes the ribosome is a larger 80S
ribosome that contains a small 40S and a
large 60S subunit.
The subunits have distinct functions in the ribosome:
The small 30S subunit is responsible for mRNA and tRNA binding. It
participates in the codon:anticidon recognition.
•It has two binding sites, the A and P sites for tRNAs.
•The P site binds the anticodon of peptidyl-tRNA (pept-tRNA) while
•The A site bind the anticodon of the aminoacyl-tRNA (aa-tRNA).
•The binding sites for mRNA and tRNA on the 30S are made of rRNA
with a very modest protein contribution.
•The 30S subunit has several morphological parts: its upper part is called
the head and the lower part the body.
•The relative movements of the head and the body are involved in
ribosomal functions, like codon-anticodon recognition.
The large 50S subunit is responsible for the catalytic
function of the ribosome: peptidyl-transfer.
•This catalytic site, the Peptidyl-Transferase Center
(PTC) is almost entirely composed of rRNA.
•This observation supports the RNA-world hypothesis
that the original ribosome was made entirely from rRNA.
Ribosome Substrates: aa-tRNAs.
tRNA molecules are very important
in protein synthesis.
tRNA role is to deliver aminoacids
to the ribosome.
Each tRNA is specific for the
delivery of a particular aminoacid.
tRNA participate in the catalysis of
peptidyl transfer (substrate assisted
catalysis).
Aminoacyl-tRNA synthetases (RSes)
•RSes catalyze the coupling of tRNA molecules to their
cognate aminoacids.
•Each of 20 RSes recognizes only one particular aminoacid
(aa) . It also recognizes a set of tRNAs cognate for this
particular aminoacid.
•Usually the recognition involves the anticodon of tRNA as it
shown in the picture for GlnRS in complex with its cognate
tRNAGln.
•The universal CCA acceptor end of tRNA is positioned in the
active site of the enzyme.
•The enzyme binds also an ATP molecule and a cognate
aminoacid in its active site.
Acylation as two sequential reactions:
•First, AA reacts with ATP:
AA + ATP => AA-AMP + PP
•Aminoacyl-adenylate is formed.
•This adenylate is retained in the active center while
the pyrophosphate PP is released from the enzyme
and degraded to the two phosphates by another
enzyme: pyro-phosphotase.
•Next, the AA is transferred from AMP to the 3'OH of
the end ribose on the CCA end of tRNA:
tRNA + AA-AMP => tRNA-AA + AMP
•As the result the aa-tRNA is formed:
•Charging of tRNAs with their corresponding AA by RS-es
represents the first major step of the genetic code deciphering.
• At this step the matching between the tRNA anticodon and the
aminoacid is established by RS.
•The second step of the decoding: the matching of the tRNA
anticodon with the mRNA codon is done by the ribosome
Acylated tRNA (AA-tRNA) is then delivered to the ribosome by elongation factor Tu.
Code Table with E.coli tRNA isoacceptors.
The ternary complex (TC) between EFTu*GTP and Cys tRNA.
To bind aa-tRNA Tu must be in GTP
form (Tu*GTP).
Tu*GDP releases aa-tRNA
Cod:AA tRNA:
Anticodon
UUU:F tF1: GAA
Cod:AA tRNA: Anticodon
UUC:F tF1: GAA
UCC:S tS5: GGA
UUA:L tL4: UAA /
NAAm
UUG:L tL5: CAA /
UCA:S tS1: UGA /
VGAm
UCG:S tS2: CGA
CUU:L tL2: GAG
CCU:P tP2: GGG
CUC:L tL2: GAG
CCC:P tP2: GGG
CUA:L tL3: UAG
CUG:L tL1: CAG
CCA:P tP3: UGG /
VGGm
CCG:P tP1: CGG
AUU:I tI1: GAU
ACU:T tT3: GGU
AUC:I tI1: GAU
ACC:T tT3: GGU
AUA:I tI2: CAU / LAU
ACA:T tT4: UGU
AUG:M tM1: CAU
ACG:T tT2: CGU
GUU:V tV2: GAC
GCU:A tA2: GGC
GUC:V tV2: GAC
GCC:A tA2: GGC
GUA:V tV1: UAC /
VAC
GUG:V tV1: UAC
/VAC
GCA:A tA1: UGC
/VACm
GCG:A tA1: UGC
/VACm
UCU:S tS5: GGA
Cod:AA tRNA:
Anticodon
UAU:Y tY1:GUA
/QUAm
UAC:Y tY1:GUA/
QUAm
UAA:Z rF1
Cod:AA tRNA:
Anticodon
UGU:C tC1: GCA
UAG:B rF1
UGG:W tW1: CCA
CAU:H tH1: GUG
/QUGm
CAC:H tH1: GUG
/QUGm
CAA:Q tQ1: UUG /SUG
CGU:R tR2: ACG (ICG)
CAG:Q tQ2: CUG
CGG:R tR3: CCG
AAU:N
QUUm
AAC:N
QUUm
AAA:K
SUUm
AAG:K
SUUm
GAU:D
QUCm
GAC:D
QUCm
GAA:E
SUCm
GAG:E
SUCm
tN1: GUU /
AGU:S tS3: GCU
tN1: GUU /
AGC:S tS3: GCU
tK1: UUU /
tK1: UUU /
AGA:R tR4: UCU /
MCUm
AGG:R tR5: CCU
tD1: GUC /
GGU:G tG3: GCC
tD1: GUC /
GGC:G tG3: GCC
tE2: UUC /
GGA:G tG2: UCC
tE2: UUC /
GGG:G tG1: CCC
UGC:C tC1: GCA
UGA:O rF2
CGC:R tR2: ACG (ICG)
CGA:R tR2: ACG (ICG)
Description of most common modified nucleotides in tRNA
V=cmo5-U (carboxymethoxy-U): increases wobbling can bind to U in the codon
L=Lysidine=k2C transforms C into a like U type that exclusively read A
Q=Queosine=complex G modification: restricted A wobbling
S=mnm5s2U (5-methylaminomethyl-2-thio-U): very restricted wobbling with U
I=deaminated A= G minus one H-bond with C can wobble with A
Elongation cycle of the ribosome
•EF-Tu*GTP*AA-tRNA complex binds to the empty Asite if the tRNA anticodon matches the codon in the Asite. This is checked by the 30S ribosomal subunit. If
the match is good the hydrolysis of GTP on EF-Tu
occurs and EF-Tu looses its affinity to the CCA end of
tRNA. This CCA end carries an aminoacid and it
moves from EF-Tu to dock in the A-site on the 50S
subunit. The ribosome to the right has the aa-tRNA
in the A site positioned there after EF-Tu has already
left.
•The 50S subunit catalyses the peptidyl transfer from
pept-tRNA in the P site to the aminoacid of aa-tRNA in
the A-site. As a result the pept-tRNA is in the A site
and the de-acylated tRNA in the P site.
•Then EF-G binds and translocates the pept-tRNA
from the A to P site. As a result the cycle is closed: we
get again the ribosome with peptidyl-tRNA in the P
site and a free A site ready to accept a new EFTu*GTP*AA-tRNA complex. However, the peptide is
one AA longer and the ribosome moves by one codon
along mRNA.
•After GTP hydrolysis, EF-G*GDP and EF-Tu*GDP
are released from the ribosome and should be
transformed back to EF-G*GTP and EF-Tu*GTP
before they can be re-used in the next cycle.
Mechanism of the peptidyl-transfer reaction
•The CCA end of the A-site aa-tRNA is fixed by
C75 interaction with G2588 of 23 rRNA.
•The CCA end of the P-site tRNA is fixed by
C74-G2285 and C75-G2284 interactions.
•This positions the attacking NH2 right on the
top of the carbonyl carbon.
•The 2’-OH of pept-tRNA is important for
peptidyl-transfer: it allows the formation of a
six-member transition state for the proton
shuffling from NH2 to the 3’-O of the sugar
(protonation of the leaving group).
Accuracy of translation
During elongation of protein synthesis the ribosome must
chouse one correct aa-tRNA isoacceptor among about 40
different aa-tRNAs competing for the same A-site on the 30S
subunit. The selection of the right aa-tRNA often depends on a
one nucleotide difference in the anticodon. Thermodynamic
difference between A:U and G:U is only about 1kcal/mol.
•Here, the Phe-tRNA anticodon, 5’-GAA, interacts with the
UUU codon for Phe in the A-site of the ribosome.
•To recognize the correct codon-anticodon base pairing the
ribosome uses bases A1493, A1492 and G530 of the 16S
rRNA of the 30S subunit to monitor the correctness of the
geometry of the codon-anticodon helix.
•The A-s and G530 make contacts with the sugars exposed
in the minor grove of codon-anticodon helix without actually
interacting with the bases. This allows the recognition of any
of the 61 right codon-anticodons.
The recognition of the third,
wobble position of the
codon by G530 is less
strict.
Ogle, J.M., et al. (2001) Recognition of cognate transfer RNA by
the 30S ribosomal subunit. Science, 292, 897-902
Activation of GTP hydrolysis on EF-Tu occurs on the recognition of cognate tRNA in A the
site: His84 of Tu becomes pushed forward by the phosphate of nucleotide A2662 of Sarcin-Ricin loop of 23S rRNA
towards the water molecule near the g-P of GTP. His84 activates water for GTP hydrolysis.
Toxin Sarcin cleaves the
loop between nucleotides
G2661 and A2662, while
another toxin, ricin, depurinates A2660, which
prevents the GTPase
activation on another
elongation factor, ER-G.
Voorhees, R.M., Schmeing, T.M., Kelley, A.C. and Ramakrishnan, V. (2010) The mechanism for activation of GTP
hydrolysis on the ribosome. Science, 330, 835-838.
tRNA accommodation in the A-site of the 50S subunit and proofreading
After GTP hydrolysis the
cognate tRNA
accommodates from “T”
into the A-site of the 50S
subunit relaxing its strained
conformation (the notion of
a “molecular spring”.) The
cognate tRNA moves to the
A-site without much
hinders.
After the accommodated into
the A-site on the 50S subunit
aa-tRNA becomes the
acceptor of peptide from the
peptidyl-tRNA in the P-site.
Schmeing, T.M., et al (2009) The Crystal
Structure of the Ribosome Bound to EF-Tu and
Aminoacyl-tRNA. Science
Occasionally non-cognate tRNA also induces GTP hydrolysis on Tu but it
is positioned slightly wrongly in the A-site. This will cause its CCA end to
move alone a different accommodation trajectory, and, secondly it is kept
only weakly on the 30S. It will therefore dissociate with a high probability
from the ribosome before reaching the A-site on the 50S subunit. This
drop-off of near cognate tRNA during its accommodation is called
“proofreading”.
Termination of protein synthesis
•Termination occurs in response to the
UAA, UAG or UGA stop codons in the A
site.
•These stop codons do not have cognate
tRNAs. Instead, they are read by protein
factors called release factors (RF).
Release factors recognize the stop
codons directly (Korostelev, 2011)
•A single release factor eRF1 in
eukaryotes recognizes all three stop
codons. The factor has a tRNA like
shape and binds to the A site of the
ribosome.
RFs have a universally conserved Gly-Gly-Gln (GGQ) sequence that needs to reach to the peptidyl-transferase center on
the 50S subunits to make the peptidyl-transferase of the 50S to run a hydrolytic reaction. Stop codon recognition may
result in the conformation change in RF that allows the accommodation of GGQ in the 50S A-site.
Korostelev, A.A. (2011) Structural aspects of translation termination on the ribosome. Rna, 17, 1409-1421
Recycling of ribosome after peptide release.
After peptide release the ribosome is left on mRNA with deacylated tRNA in the P-site and a release factor RF in
the A site.
In prokaryotes this stable ribosomal state is disassembled in the following way (Karimi et al., 1999) :
•First, RF1 is removed by release factor
RF3 in the reaction that requires GTP
hydrolysis.
•Then the ribosome is opened
(dissociated into subunits) by a
concerted action of EF-G*GTP and the
ribosome recycling factor RRF.
•As a result the mRNA and deacylated
tRNA remains bound to the 30S subunit.
•Deacylated tRNA is then removed by
initiation factor IF3 and this destabilizes
mRNA binding.
•mRNA leaves and the 30S subunit is
now ready to initiate a new cycle of
protein synthesis.
In eukaryotes the complex eRF1*eRF3*GTP recognizes the stop codon in the A-site and promote hydrolysis of
the ester bond of the peptidyl-tRNA by the ribosome to release the nascent peptide. The mechanism of ribosomal
recycling in eukaryotes is not known.
Initiation of protein synthesis in prokaryotes
Specialized initiator tRNA.
All tRNAs look very similar but there is a special tRNA that is only involved in the initiation of protein synthesis.
Several specific universal feature of this tRNAiMet conserved in all initiator tRNAs from bacteria to human are:
•The presence of the three consecutive GC base pairs in its anticodon stem (the middle tRNA).
•The absence of terminal base-pair in bacteria and a weak A:U base-pair in eukaryotes.
•The unpaired base pair on the top of the acceptor stem is important for Met-tRNAi formylation in bacteria which is in turn
important for fMet-tRNAi interaction with the initiation factor IF2.
•Three initiation factors IF1, IF2, IF3 and the initiator tRNA participate in initiation in prokaryotes.
•The major role of the initiation factors is to select the initiator fMet-tRNAi into the P-site of the 30S pre-initiation complex
and then into the 70S initiation complex.
•Besides, IF3 removes deacylated tRNA from the 30S subunit after the termination.
Initiation complex formation in prokaryotes
•At the initiation stage IF3 assures that only the initiator
tRNA binds to the IF2:IF3*30S*mRNA complex.
•IF3 binding to 30S affects the 30S conformation in such
a way that its P-site widens and becomes more
accessible for all tRNAs.
•The 30S subunit in this altered conformation interacts
with G:C-base-pairs of fMet-tRNAi effectively selecting it
over other tRNAs.
•IF1 binding to the 30S and blocks the A site of the 30S
preventing tRNA binding to the A site of 30S during
initiation.
•IF2 binds with high affinity to the 30S and to formylated
tRNAs and effectively selects fMet-tRNAi over all other
tRNAs (which are acylated but not formylated) in the 30S
pre-initiation complex (PIC).
•IF2 has a double role in initiation. First, it selects fMettRNAi into the 30S PIC and, second, IF2 promotes fast
docking of the 50S subunit to the
IF1:IF3:30S:mRNA:fMet-tRNA complex but only when it
contains fMet-tRNA.
•The docking of 50S results in GTP hydrolysis on IF2
and its release from the 70S ribosome. Now the ribosome
is ready to enter the elongation cycle.
mRNA binding in bacterial initiation
•The binding of mRNA to the 30S subunit usually occurs through a Shine Dalgarno (SD) sequence of mRNA.
•The SD has a UAAGGAGGU consensus complementary to the very 3' end of the 16S rRNA of the 30S subunit.
•mRNA binding is an intrinsic property of prokaryotic ribosomes.
•Initiation then occurs on the first AUG 3' from the SD sequence. This AUG is selected by fMet-tRNA bound to the 30S
•Many mRNAs have only a short version of the SD like AAGGA but this is usually sufficient for its proper binding to the
30S subunit.
•The initiator tRNA determines the reading frame of the mRNA by selecting the AUG initiator codon into the
P site through its perfect complementarily to fMet-tRNAi anti-codon.
•The SD sequence is usually close to the 5' end of mRNA and the ribosome binds to it right after it has been
synthesized by RNAP.
•The first ribosome follows RNAP very tightly and other ribosomes can load on the same mRNA forming a train of
ribosomes that follows the polymerase. This ribosomal train is called a polysome. A tight coupling between
transcription and translation in prokaryotes explains why such simple trick as the SD sequence would work.
Translation initiation in eukaryotes: four major stages of initiation.
Translation initiation in eukaryotes is a relatively well understood despite a large number of factors participating in it.
Four Main Stages of Initiation:
I.) 43S complex includes 40S ribosomal subunit plus
initiation factors eIF1, eIF1A, eIF3 and the
eIF2*GTP*Met-tRNAi complex. It most probably
includes eIF5 and eIF5B as well. eIF2 brings MettRNAi into the initiation complex.
2.) Formation of the complex between mRNA and the
initiation factor IF4F. eIF4F is composed of 4E, 4G
and 4A.
3.) The binding of eIF4F:mRNA to the 43S complex through
the interaction between eIF4G and eIF3 is followed
by the scanning by the 43S complex along mRNA
until Met-tRNAi recognizes the AUG codon. This
promotes hydrolysis of GTP on eIF2 which requires
eIF5. eIF2 leaves and Met-tRNAi interacts now with
eIF5B. The net result is the formation of a 48S
initiation complex with mRNA and Met-tRNA
properly position on the 40S subunit.
4.) After the binding of Met-tRNAi to eIF5B the mature 48S
complex docks to the 60S subunit. GTP hydrolysis
on eIF5B results in its leaving the 80S and in the
formation of a functional 80S initiation complex
committed to elongation. Another factor, eIF5A may
participate in the formation of the first peptide bond.
Regulation of initiation by eIF2 phosphorylation
The major role of eIF2 is the delivery of Met-tRNAi to the P-site of the 40S subunit. In this, it plays a role of a functional
analogue of eIEF1 or EF-Tu except that the elongation factor delivers aa-tRNAs to the A-site of the 80S ribosome.
•eIF2 is regulated by phosphorylation of Ser51 in
eIF2a subunit. The real target of eIF2a
phosphorylation is the GDP/GTP exchange factor
eIF2B which forms a tight complex with the
phosphorylated eIF2*GDP.
•This results in the sequestration of eIF2B which is
present in the cell in much lower amounts than eIF2
into eIF2B:P-eIF2 complexes.
•In the absence of free eIF2B no GTP/GDP
exchange on eIF2*GDP occurs and the initiation of
translation stops.
Four kinases acting on eIF2
They phosphorylate eIF2 on Ser51:
•HRI kinase (Heme Regulated Inhibitor)
that phosphorylates IF2 in reticulocites.
•PKR is activated by interferon and also
in the presence of double stranded RNA.
Dever, T.E. (1999) Translation initiation: adept at adapting.
Trends Biochem Sci, 24, 398-403
mRNA recruitment to the 40S subunit
•eIF4F consists of eIF4G, eIF4E and eIF4A.
•The scaffold factor eIF4G plays a central role by presenting
binding sites for eIF4E and eIF4A and for a number of other
factors.
•eIF4G can be subdivided into the N-terminal part, middle
part (M) and C-terminal part.
•Binding sites for different proteins are distributed in a linear
manner along the eIF4G sequence:
The role of eIF4E in the binding of the 5’-cap structure
•After the delivery of mRNA into the cytoplasm initiation factor eIF4E
binds to the m7GpppG- cap structure of the mRNA replacing the
nuclear Cap-binding factor. eIF4E itself is a subunit of eIF4F.
•eIF4E binds m7GpppG in the cleft formed by several hydrophobic Trp
(W) residues of the factor as shown below. These Trp residues play
crucial role in the binding of a hydrophobic 7mG base.
•G base of m7G is recognized by E103 while basic Arg112, Arg157
and Lys 162 stabilize the binding of the tri-phosphate moiety of the cap
structure
Gingras, A.C., Raught, B. and Sonenberg, N. (1999) eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of
translation. Annu Rev Biochem, 68, 913-963
Regulation of mRNA delivery to the 40S subunit (1)
•The binding affinity of capped mRNA is also increased by
phosphorylation of Ser 209 in eIF4E.
•The structural explanation of this effect is that the P-Ser209 may
form a salt bridge with Lys159 which would form an ark over mRNA
trapping mRNA in the binding cleft of eIF4E.
•Phosphorylation of See209 is promoted by MNK1 kinase that
has its binding site on the eIF4G scaffold protein
The binding site of eIF4E for eIF4G is on the side of eIF4E opposite to the
cap-binding site.
This site binds non-structured peptides of defined composition similar to that
forming the binding site on eIF4G.
Several different cell proteins called 4E binding protein (4E-BP) bind to
eIF4E with a high affinity and regulate its binding to the adaptor eIF4G in a
competitive manner.
Gingras, A.C., Raught, B. and Sonenberg, N. (1999) eIF4 initiation factors:
effectors of mRNA recruitment to ribosomes and regulators of translation.
Annu Rev Biochem, 68, 913-963
Regulation of mRNA delivery to the 40S subunit (2)
•The binding of 4E-BPs (there are several of those; they are
small proteins with MW around 12 kD) is regulated by
phosphorylation.
•Phosphorylation of 4E-BPs stimulates the translation of
capped mRNAs by releasing eIF4E from inhibitory
complexes with 4E-BPs.
•In case of some virus infections ( EMCV is an example) the
virus can shut down the translation of capped mRNAs by
de-phosphorylating 4E-BP by virus encoded phosphatases.
Translation regulation in the cell
Gingras, A.C., Raught, B. and Sonenberg, N. (1999) eIF4 initiation factors:
effectors of mRNA recruitment to ribosomes and regulators of translation.
Annu Rev Biochem, 68, 913-963
Internal Ribosome Entry Sites (IRES)
Most cell mRNAs are translated through a conventional mechanism where eIF4F binds to the
cap structure in the mRNA.
Some viruses have very long and exquisitely structured 5' UTR (Un-Translated Region) in their mRNAs but no cap
structures. It has been found that they use Internal Ribosome Entry Sites (IRES) to initiate their translation.
IRES of FMDV (Foot-and Mouth Disease Virus) consists of 300-400 nt long highly structured 5’ UTR preceding the
first initiation codon. IRES can bind directly to eIF4G and does not require eIF4E:
The host translation of capped mRNAs is blocked by FMDV due to eIF4G cleavage, since
the cap-binding moiety of eIF4F is now physically separated from its eIF3-binding moiety.
miRNAs and siRNAs in post-transcriptional mRNA silencing
miRNAs (micro-RNAs) regulate gene
expression at post-transcriptional level.
Hundreds of miRNA have been recently
identified in human genome and genomes
of other organisms.
•Pre-miRNA is first processed in the
nucleus by the Drosha/Pasha
(DGCR8=Pasha) nuclease (RNase III). The
place of the cleavage of pri-miRNA hairpins
from pre-miRNA precursor is determined
by Pasha/DGCR8 (DiGeorge syndrome
critical region gene 8). Pasha binds at the
boundary between dsRNA and ssRNA in
pri-miRNA.
•Then the resulting dsRNA hairpin is
exported to the cytoplasm by Exportine 5.
•In the cytoplasm dsRNA hairpins are
cleaved by Dicer nuclease into 21-23 bp
miRNA duplexes.
•Dicer recognizes the ends of pre-miRNA
hairpin produced by the Microprocessor
Drosha-Pasha complex (a 3’ two
nucleotide overhang) using its PAZ
domain.
MacRae, I.J. and Doudna, J.A. (2007) Ribonuclease revisited:
structural insights into ribonuclease III family enzymes. Curr Opin
Struct Biol, 17, 138-145
Kim, V.N., Han, J. and Siomi, M.C. (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol, 10, 126-139
Exportin 5 recognizes 3’-overnang in (micro) procesed pre-miRNA
Guttler, T. and Gorlich, D. (2011) Ran-dependent nuclear export mediators: a structural perspective. Embo J, 30, 3457-3474
Ago is a core of RISC: There are 8 different Ago proteins in humans. RISC complex type is determined
by Ago protein: the target mRNA may either be cleaved by RISC or just translationally silenced.
Jinek, M. and Doudna, J.A. (2009) A three-dimensional view of the molecular machinery of RNA interference. Nature, 457, 405-412
Models of translation inhibition by RISC
AGO can interact with a number of proteins.
In P-bodies it interacts with GW182 protein that interacts with PABP.
This interaction recruits poly-A degrading complex NOT-CCR4-CAF1.
GW (Gly-Trp) repeats interact with AGO. M and C-domain participate
in silencing. Binding of M/C to PABP interferes with its binding to
eIF4G blocking the close loop formation, allowing de-adenylation.
Tritschler, F., Huntzinger, E. and Izaurralde, E. (2010) Role of GW182 proteins and PABPC1 in
the miRNA pathway: a sense of deja vu. Nat Rev Mol Cell Biol, 11, 379-384
Degradation of eukaryotic mRNAs is promoted by decapping factors, by the removal of polyA tail and by
endonucleolytic cleavage.
•Initial deadenylation is carried out by PARN or CCR4NOT-CAF1 complex. After the removal of the poly-A tail,
Exosome takes over mRNA degradation from 3’ end.
•For 5’=>3’decay the cap is removed by DCP1-DCP2
complex which also stimulated by Lsm1-7 complex bound
at 3’ end (in high eukaryotes) or by Edc3 complex (in low
eukaryotes). De-capping is then followed by XRN1
exonuclealse degradation. DspS degrades the Cap
structure.
•Endonucleolitic cleavage of mRNA is unusual in
eukaryotes. It is performed by mRNA- specific
endonucleases.
•mRNA degradation is also promoted by the presence of
AU sequences in the 3’ UTR of mRNAs.
Garneau, N.L., Wilusz, J. and Wilusz, C.J. (2007) The highways and byways of mRNA decay. Nat Rev Mol Cell Biol, 8, 113-126
Simplified scheme of NMD
More detailed scheme of NMD
mRNAs of non-functional proteins are eliminated by NMD
Alternative splicing often generates mRNAs for non-functional
proteins with premature stop codons close to the beginning of
mRNA (as in the Sxl and Tra mRNAs in male flies discussed
earlier). The cell uses an NMD mechanism to quickly destroy
those mRNAs in the cytoplasm before much of non-functional
protein is produced. The NMD is based on the fact that
normally mRNAs have stop codons in their last exons.
During the first translation pass the ribosome displaces
nuclear mRNP proteins including proteins composing EJC.
The displaced EJC proteins return then back to the nucleus.
•NMD results in the destruction of mRNAs that
have a Premature Termination Codon (PTC) more
than 50 nucleotides upstream of the exon-exon
junction containing EJC.
•Additional Upf1 and Upf2 factors are recruited in
the cytoplasm.
•Release factor (RF) forms a bridging structure
consisting of Upf proteins ending at EJC.
•Such a complex between release factors (RF),
Upf-s and EJC promotes the NMD response.
•NMD requires SMG1 kinase and SMG7 scaffold
protein binding. SMG7 is an adaptor protein that
promotes DCP1/2 complex binding and cap
removal.
NMD is used to regulate gene expression of some splicing factors.
Lejeune, F. and Maquat, L.E. (2005) Mechanistic links between nonsense-mediated mRNA decay and pre-mRNA splicing in mammalian cells. Curr Opin Cell Biol, 17, 309-315
Selected References on Ribosomal Accuracy
•Johansson, M., Lovmar, M. and Ehrenberg, M. (2008) Rate and accuracy of bacterial protein synthesis revisited. Curr
Opin Microbiol, 11, 141-147.
•Karimi, R., Pavlov, M.Y., Buckingham, R.H. and Ehrenberg, M. (1999) Novel roles for classical factors at the interface
between translation termination and initiation. Mol Cell, 3, 601-609.
•Korostelev, A.A. (2011) Structural aspects of translation termination on the ribosome. Rna, 17, 1409-1421.
•Liljas, A., Liljas, L., Piskur, J., Lindblom, G., Nissen, P. and Kjeldgaard, M. (2009) Textbook of Structural Biology. World
Scientific Publishing Co. Pte.Ltd, Singapur.
•Ogle, J.M., Brodersen, D.E., Clemons, W.M., Jr., Tarry, M.J., Carter, A.P. and Ramakrishnan, V. (2001) Recognition of
cognate transfer RNA by the 30S ribosomal subunit. Science, 292, 897-902.
•Ogle, J.M., Carter, A.P. and Ramakrishnan, V. (2003) Insights into the decoding mechanism from recent ribosome
structures. Trends Biochem Sci, 28, 259-266.
•Ogle, J.M. and Ramakrishnan, V. (2005) Structural insights into translational fidelity. Annu Rev Biochem, 74, 129-177.
•Ramakrishnan, V. (2008) What we have learned from ribosome structures. Biochem Soc Trans, 36, 567-574.
•Schmeing, T.M., Voorhees, R.M., Kelley, A.C., Gao, Y.G., Murphy, F.V.t., Weir, J.R. and Ramakrishnan, V. (2009) The
Crystal Structure of the Ribosome Bound to EF-Tu and Aminoacyl-tRNA. Science.
•Schmeing, T.M., Voorhees, R.M., Kelley, A.C. and Ramakrishnan, V. (2011) How mutations in tRNA distant from the
anticodon affect the fidelity of decoding. Nat Struct Mol Biol, 18, 432-436.
•Schuette, J.C., Murphy, F.V.t., Kelley, A.C., Weir, J.R., Giesebrecht, J., Connell, S.R., Loerke, J., Mielke, T., Zhang,
W., Penczek, P.A., Ramakrishnan, V. and Spahn, C.M. (2009) GTPase activation of elongation factor EF-Tu by the
ribosome during decoding. Embo J, 28, 755-765.
•Voorhees, R.M., Schmeing, T.M., Kelley, A.C. and Ramakrishnan, V. (2010) The mechanism for activation of GTP
hydrolysis on the ribosome. Science, 330, 835-838.
•Wohlgemuth, I., Pohl, C., Mittelstaet, J., Konevega, A.L. and Rodnina, M.V. (2011) Evolutionary optimization of speed
and accuracy of decoding on the ribosome. Philos Trans R Soc Lond B Biol Sci, 366, 2979-2986.