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I N Shatsky

Publications and source records attributed to I N Shatsky.

At least 19 recordsLinked to original sources
PubMed2001

Molecular mechanisms of translation initiation in eukaryotes.

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5' end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.

Amino Acid Sequence↗
PubMed2001

Lipoxygenase mRNA silencing in erythroid differentiation: The 3'UTR regulatory complex controls 60S ribosomal subunit joining.

15-lipoxygenase (LOX) expression is translationally silenced in early erythroid precursor cells by a specific mRNA-protein complex formed between the differentiation control element in the 3' untranslated region (UTR) and hnRNPs K and E1. The 3'UTR regulatory complex prevents translation initiation by an unknown mechanism. We demonstrate that the 40S ribosomal subunit can be recruited and scan to the translation initiation codon even when the silencing complex is bound to the 3'UTR. However, the joining of the 60S ribosomal subunit at the AUG codon to form a translation competent 80S ribosome is inhibited, unless initiation is mediated by the IGR-IRES of the cricket paralysis virus. These findings identify the critical step at which LOX mRNA translation is controlled and reveal that 60S subunit joining can be specifically regulated.

3' Untranslated Regions↗
PubMed1998

Translation eukaryotic initiation factor 4G recognizes a specific structural element within the internal ribosome entry site of encephalomyocarditis virus RNA.

A complex of eukaryotic initiation factors (eIFs) 4A, 4E, and 4G (collectively termed eIF4F) plays a key role in recruiting mRNAs to ribosomes during translation initiation. The site of ribosomal entry onto most mRNAs is determined by interaction of the 5'-terminal cap with eIF4E; eIFs 4A and 4G may facilitate ribosomal entry by modifying mRNA structure near the cap and by interacting with ribosome-associated factors. eIF4G recruits uncapped encephalomyocarditis virus (EMCV) mRNA to ribosomes without the involvement of eIF4E by binding directly to the approximately 450-nucleotide long EMCV internal ribosome entry site (IRES). We have used chemical and enzymatic probing to map the eIF4G binding site to a structural element within the J-K domain of the EMCV IRES that consists of an oligo(A) loop at the junction of three helices. The oligo(A) loop itself is not sufficient to form stable complexes with eIF4G since alteration of its structural context abolished its interaction with eIF4G. Addition of wild type or trans-dominant mutant forms of eIF4A to binary IRES.eIF4G complexes did not further alter the pattern of chemical/enzymatic modification of the IRES.

Animals↗
PubMed1998

A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs.

Initiation of translation of hepatitis C virus and classical swine fever virus mRNAs results from internal ribosomal entry. We reconstituted internal ribosomal entry in vitro from purified translation components and monitored assembly of 48S ribosomal preinitiation complexes by toe-printing. Ribosomal subunits (40S) formed stable binary complexes on both mRNAs. The complex structure of these RNAs determined the correct positioning of the initiation codon in the ribosomal "P" site in binary complexes. Ribosomal binding and positioning on these mRNAs did not require the initiation factors eIF3, eIF4A, eIF4B, and eIF4F and translation of these mRNAs was not inhibited by a trans-dominant eIF4A mutant. Addition of Met-tRNAiMet, eIF2, and GTP to these binary ribosomal complexes resulted in formation of 48S preinitiation complexes. The striking similarities between this eukaryotic initiation mechanism and the mechanism of translation initiation in prokaryotes are discussed.

Animals↗
PubMed1998

Specific interaction of eukaryotic translation initiation factor 3 with the 5' nontranslated regions of hepatitis C virus and classical swine fever virus RNAs.

Translation of hepatitis C virus (HCV) and classical swine fever virus (CSFV) RNAs is initiated by cap-independent attachment (internal entry) of ribosomes to the approximately 350-nucleotide internal ribosomal entry segment (IRES) at the 5' end of both RNAs. Eukaryotic initiation factor 3 (eIF3) binds specifically to HCV and CSFV IRESs and plays an essential role in the initiation process on them. Here we report the results of chemical and enzymatic footprinting analyses of binary eIF3-IRES complexes, which have been used to identify the eIF3 binding sites on HCV and CSFV IRESs. eIF3 protected an internal bulge in the apical stem IIIb of domain III of the CSFV IRES from chemical modification and protected bonds in and adjacent to this bulge from cleavage by RNases ONE and V1. eIF3 protected an analagous region in domain III of the HCV IRES from cleavage by these enzymes. These results are consistent with the results of primer extension analyses and were supported by observations that deletion of stem-loop IIIb or of the adjacent hairpin IIIc from the HCV IRES abrogated the binding of eIF3 to this RNA. This is the first report that eIF3 is able to bind a eukaryotic mRNA in a sequence- or structure-specific manner. UV cross-linking of eIF3 to [32P]UTP-labelled HCV and CSFV IRES elements resulted in strong labelling of 4 (p170, p116, p66, and p47) of the 10 subunits of eIF3, 1 or more of which are likely to be determinants of these interactions. In the cytoplasm, eIF3 is stoichiometrically associated with free 40S ribosomal subunits. The results presented here are consistent with a model in which binding of these two translation components to separate, specific sites on both HCV and CSFV IRESs enhances the efficiency and accuracy of binding of these RNAs to 40S subunits in an orientation that promotes entry of the initiation codon into the ribosomal P site.

Animals↗
PubMed1996

Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry.

Translation of picornavirus RNA is initiated after ribosomal binding to an internal ribosomal entry site (IRES) within the 5' untranslated region. We have reconstituted IRES-mediated initiation on encephalomyocarditis virus RNA from purified components and used primer extension analysis to confirm the fidelity of 48S preinitiation complex formation. Eukaryotic initiation factor 2 (eIF2), eIF3, and eIF4F were required for initiation; eIF4B and to a lesser extent the pyrimidine tract-binding protein stimulated this process. We show that eIF4F binds to the IRES in a novel cap-independent manner and suggest that cap- and IRES-dependent initiation mechanisms utilize different modes of interaction with this factor to promote ribosomal attachment to mRNA.

Base Sequence↗
PubMed1996

Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes.

Eukaryotic translation is initiated following binding of ribosomes either to the capped 5' end of an mRNA or to an internal ribosomal entry site (IRES) within its 5' nontranslated region. These processes are both mediated by eukaryotic initiation factor 4F (eIF4F), which consists of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G subunits. Here we present a functional analysis of eIF4F which defines the subunits and subunit domains necessary for its function in initiation mediated by the prototypical IRES element of encephalomyocarditis virus. In an initiation reaction reconstituted in vitro from purified translation components and lacking eIF4A and -4F, IRES-mediated initiation did not require the cap-binding protein eIF4E but was absolutely dependent on eIF4A and the central third of eIF4G. This central domain of eIF4G bound strongly and specifically to a structural element within the encephalomyocarditis virus IRES upstream of the initiation codon in an ATP-independent manner and with the same specificity as eIF4F. The carboxy-terminal third of eIF4G did not bind to the IRES. The central domain of eIF4G was itself UV cross-linked to the IRES and strongly stimulated UV cross-linking of eIF4A to the IRES in conjunction with either eIF4B or with the carboxy-terminal third of eIF4G.

Encephalomyocarditis virus↗
PubMed1996

Structural analysis of the interaction of the pyrimidine tract-binding protein with the internal ribosomal entry site of encephalomyocarditis virus and foot-and-mouth disease virus RNAs.

Initiation of translation of a subset of eukaryotic mRNAs results from internal ribosomal entry. This process is exemplified by encephalomyocarditis virus (EMCV), which contains an internal ribosomal entry site (IRES) within its 5' nontranslated region that is approximately 450-nt long and consists of a series of stem-loops designated H-L. We have previously identified a cellular 58-kDa polypeptide that binds specifically to this IRES and that is implicated in its function as the pyrimidine tract-binding protein PTB. We have now mapped PTB binding sites directly on the IRES elements of EMCV and the related foot-and-mouth disease virus (FMDV) using structure-specific enzymatic probes and base-specific chemical probes. PTB bound to six sites on the EMCV IRES: site 1 (UCUU401) is upstream of domain H, site 2 is the basal helix of domain H (nt 407-410 and 440-443), site 3 (UCUUU423) is the apical loop of domain H, site 4 is the apical helix and adjacent internal bulged loop of domain K, site 5 (CUUUA750) is the apical loop of domain K, and site 6 (CCUUU815) is downstream of domain L. PTB bound to sites on the FMDV IRES that correspond precisely to EMCV sites 3, 5, and 6. These sites have the consensus sequence CUUU and form two groups that are located near to the 5' and 3' borders of these IRES elements. Their position, and the effects of mutation of them on IRES function are consistent with PTB's role in IRES-mediated initiation being to bind to multiple sites in the IRES, thereby stabilizing a specific active conformation.

Aphthovirus↗
PubMed1994

The RNA-binding properties of protein synthesis initiation factor eIF-2.

Protein synthesis initiation factor eIF-2 bound ATP in the presence or absence of Mg2+ ions. ATP impaired the binding of GTP or GDP to eIF-2. However, excess GTP did not significantly decrease the binding of ATP to eIF-2, suggesting eIF-2 has distinct ATP and GTP binding sites. Highly purified eIF-2 can bind mRNA, and this did not require the mRNA to be capped. mRNA binding was saturable, and maximal binding corresponded to about 0.4 mol mRNA bound per mol eIF-2. GTP, and, at lower concentrations, GDP, inhibited the binding of mRNA to eIF-2. In addition, ATP and other nucleoside triphosphates decreased mRNA binding. The implications of these findings for the structure and function of eIF-2 are discussed. Preparations of eIF-2 deficient in the beta-subunit showed reduced ability to bind mRNA, suggesting that while it is not essential for mRNA binding, this subunit is involved in the interaction. Consistent with this is the observation that ultraviolet crosslinking of mRNA to eIF-2 resulted primarily in labelling of the beta-subunit. Subsequent analysis revealed that mRNA was cross-linked to the C-terminal region of eIF-2b which contains a putative Zn-finger structure.

Adenosine Triphosphate↗
PubMed1992

Unusual ribosome binding properties of mRNA encoding bacteriophage lambda repressor.

The mRNA encoding repressor cI of phage lambda is the only known E. coli message which starts directly with the initiation AUG codon. The ability of in vitro synthesized cI mRNA fragments (150 or 400 nts) to form ternary initiation complexes has been studied using the toeprint method. In the presence of tRNA(Met)f, these fragments are capable of forming the ternary complexes at the 5'-terminal AUG codon not only with 30S subunits but also with undissociated 70S ribosomes (70S tight couples). In the latter case, no binding at other positions of cI mRNA can be detected at all. The starting region of cI mRNA has a single stranded conformation and is highly enriched in A-residues. This feature of cI mRNA RBS is suggested to be the main factor which allows cI mRNA to form the initiation complex with the ribosome. Unlike 30S subunits, the binding to 70S tight couples is not affected by any of the initiation factors, although it is as efficient as that to 30S subunits supplemented with the factors. 30S subunits prefer to associate with the internal RBSs of the preformed mRNA molecules, provided that they are not sequestered by the secondary structure. In contrast, 70S tight couples tend to avoid extra sequences upstream of the codon directed to the P site and occupy a position as close as possible to the 5'-end of the message. This has been found to be the case both for tRNA(Met)f and for elongator tRNA(Glu)2. The structural features of mRNA RBSs which influence their different binding for 30S subunits and 70S ribosomes are discussed.

Bacteriophage lambda↗
PubMed1991

RNA--protein interactions within the internal translation initiation region of encephalomyocarditis virus RNA.

Various derivatives of the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) RNA have been used to analyze by UV-cross-linking its interaction with mRNA binding proteins from ascites carcinoma Krebs-2 cells. A doublet of proteins with Mr 58 and 60 kD bound to two regions of the IRES. One site is centered at nt 420-421 of EMCV RNA whereas the other is located between nt 315-377. Both sites form hairpin structures, the loops of which contain UCUUU motif, conserved among cardio- and aphthoviruses. The interaction of p58 and p60 with IRES is affected by the integrity of the stem-loop structure proximal to the start AUG codon (nts 680-787), although, under similar conditions, cross-linking of these proteins to this region was not detected. Deletions in the main recognition site of p58 strongly reduce the initiation activity of the IRES in vitro. However, elimination of p58 (p60) binding by these mutations does not completely abolish the ability of the IRES to direct polypeptide synthesis starting from the authentic AUG codon. The IRES can be assembled in vitro from two covalently unlinked transcripts, one containing the target site for p58 and the other encompassing the remaining part of the IRES fused to a reporter gene, resulting in considerable restoration of its activity. Implications of these findings for the mechanism of initiation resulting from internal entry of ribosomes are discussed.

Animals↗
PubMed1991

Spatial organization of template polynucleotides on the ribosome determined by fluorescence methods.

The spatial organization of template polynucleotides on the ribosome and the dynamics of their interaction with 30 S subunits have been studied by fluorescence spectroscopy. The topography of the mRNA in the ribosome has been determined using singlet-singlet energy transfer. This method has allowed us to estimate distances between donors and acceptors of energy which have been linked to the terminal residues of template polynucleotides (poly- and oligo(U) and oligo(A] and 16 S RNA or to SH-groups of ribosomal proteins S1 and S8. The dynamics of mRNA-ribosome interaction have been investigated by the fluorescence stopped-flow technique. It has been shown that the binding to the 30 S subunit of poly(U) with length much shorter (16 nucleotides) than that covered by the ribosome is greatly enhanced by protein S1. However, the final position of oligo(U)16 on the 30 S subunit, which probably includes the ribosomal decoding site, proves to be quite different from that occupied by oligo(U)16 on a free protein S1. Interaction of oligo- and poly(U) with the 30 S subunit occurs in at least two steps: the first one is as fast as the interaction of poly(U) with free S1, whereas the second step represents a first-order reaction. Therefore, the second step may reflect some rearrangement of the template in the ribosome after its primary binding. It is suggested that protein S1 in some cases may fulfill the role of a transient binding site for mRNA in the course of its interaction with the ribosome. The general shape of the template in the mRNA binding region of the ribosome has been studied using various synthetic ribopolynucleotides and has been shown to be similar. It can be represented by a loop(s) or "U-turn(s)". On the basis of estimation of distances from the ends of poly(U) to some well-localized points on the 30 S ribosomal surface, a tentative model of mRNA path through the ribosome is proposed.

Bacterial Proteins↗
PubMed1991

A complex RNA sequence determines the internal initiation of encephalomyocarditis virus RNA translation.

Translation initiation on EMCV RNA occurs via binding of ribosomes to an internal sequence within the 5' noncoding region. To investigate the organization of the internal ribosome entry site (IRES) we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of EMCV RNA. Three functional regions have been distinguished: a sequence between nts 315-484 and the upper parts of the double-helical structural domains III (nts 488-647) and IV (nts 701-763). The first one greatly enhances translation, but is not absolutely necessary for internal initiation. The other two regions are indispensable to this process. A sequence within domain IV determines inhibition of in vitro translation of mRNAs with 5'-terminal dependent initiation. It is proposed to interact with a translational factor(s) common to the internal and 5'-terminal dependent initiation.

Binding Sites↗
PubMed1990

A factor that specifically binds to the 5'-untranslated region of encephalomyocarditis virus RNA.

A protein factor that specifically binds to the 5'-untranslated region of encephalomyocarditis virus (EMCV) RNA has been found in extracts of ascites carcinoma Krebs-2 cells. This was done using UV-irradiation on extracts supplemented with in vitro synthesized 32P-labelled transcripts followed by analysis of crosslinked proteins by SDS-polyacrylamide gel electrophoresis. The transcripts represented the viral RNA sequence from nt 315 to 1155, its derivatives with internal deletions or truncated forms. This set of transcripts has allowed us to find out that the factor (p58) binds to EMCV RNA within the sequence 315-485.

Animals↗
PubMed1985

Topography of RNA in the ribosome: location of the 5 S RNA residues A39 and U40 on the central protuberance of the 50 S subunit.

The internal site of 5 S RNA comprising residues A39 and U40 has been localized on the E. coli 50 S ribosomal subunit by immune electron microscopy. It has been found to be located on the interface side of the central protuberance at the position distinctly apart but very close to the position of the 5 S RNA 3'-end providing evidence for a quite compact folded conformation of the 5 S RNA in situ.

Centrifugation, Density Gradient↗
PubMed1983

Localization of 5' and 3' ends of the ribosome-bound segment of template polynucleotides by immune electron microscopy.

Poly(U) with an average chain length of 40-70 nucleotides was modified at the 5'- or 3'-terminal residues with 2,4-dinitrophenyl derivatives. The modified poly(U) was used to form 30S.poly(U) or 70S.poly(U).Phe-tRNA complexes. Localization of the 5' and 3' ends of the template polynucleotide on the 30S subunit and the 70S ribosome was performed by immune electron microscopy using antibodies against dinitrophenyl haptens. The 5' and 3' ends of poly(U) (putative entry and exit sites of the message) were found in the same region both on the 30S subunit and the 70S ribosome. They were located on the dorsal side of the 30S subunit between the head and the body near the groove bordering the side ledge (platform). Comparison of the size of this region with the possible length of the polynucleotide chain covered by the ribosome allowed us to suggest that the message makes a 'U-turn" (or forms a 'loop') as it passes through the ribosome.

Binding Sites↗