* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download AA-tRNA - Studentportalen
Survey
Document related concepts
Cell nucleus wikipedia , lookup
Phosphorylation wikipedia , lookup
Signal transduction wikipedia , lookup
Protein phosphorylation wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Intrinsically disordered proteins wikipedia , lookup
Cooperative binding wikipedia , lookup
List of types of proteins wikipedia , lookup
Gene expression wikipedia , lookup
Biosynthesis wikipedia , lookup
Transcript
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.