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1.
RNA unwinding in translation: assembly of helicase complex intermediates comprising eukaryotic initiation factors eIF-4F and eIF-4B. 总被引:19,自引:6,他引:13
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Ribosome binding to mRNA requires the concerted action of three initiation factors, eIF-4A, eIF-4B, and eIF-4F, and the hydrolysis of ATP in a mechanism that is not well understood. Several lines of evidence support a model by which these factors bind to the 5' end of mRNA and unwind proximal secondary structure, thus allowing 40S ribosomal subunits to bind. We have previously used an unwinding assay to demonstrate that eIF-4A or eIF-4F in combination with eIF-4B functions as an RNA helicase. To elucidate the molecular mechanism of RNA unwinding, we used a mobility shift electrophoresis assay which allows the simultaneous analysis of unwinding and complex formation between these factors and RNA. eIF-4F forms a stable complex (complex A) with duplex RNA in the absence of ATP. Addition of eIF-4B results in the formation of a second complex (complex B) of slower mobility in the gel. In the presence of ATP, both complexes dissociate, concomitant with the unwinding of the duplex RNA. We present evidence to suggest that unwinding occurs in a processive as opposed to distributive manner. Thus, we conclude that helicase complexes that are formed in the absence of ATP on duplex RNA translocate processively along the RNA in an ATP-dependent reaction and melt secondary structure. These helicase complexes therefore represent intermediates in the unwinding process of mRNA that could precede ribosome binding. 相似文献
2.
The technique of primer extension inhibition has been adapted to analyze the eukaryotic ribosome-mRNA interaction. Formation of the ribosome-mRNA complex was performed in a nuclease-treated rabbit reticulocyte lysate. Before primer extension analysis, however, the complex is isolated by sucrose gradient centrifugation. Both 80 S- and 40 S-mRNA complexes can be individually analyzed because of this isolation step. 80 S ribosomes and 40 S ribosomal subunits could be localized at the initiation codon by a number of independent means where all complexes were formed in a manner consistent with the current understanding of the initiation pathway for translation in eukaryotes. Complexes were also isolated with the aid of the antibiotic edeine, where the 40 S ribosomal subunit was not located at the initiation codon, but 5' to the initiation codon. This extension inhibition assay was used to complement studies regarding the ATP dependence of the 40 S-mRNA interacting initiation steps that involve the mammalian RNA-interacting initiation factors eIF-4A, -4B, and -4F. A strong requirement for ATP was observed for 40 S-mRNA complex formation. A factor-mediated stimulation of complex formation by a combination of eIF-4A, -4B, and -4F was observed, and was one which required the presence of ATP. This factor-mediated ATP-dependent stimulation of complex formation was significantly inhibited by preincubating eIF-4A with the ATP analog 5'-p-fluorosulfonylbenzoyl adenosine. Finally, all complexes accumulated to a significant degree were analyzed by the primer extension assay. It was found that the 40 S ribosomal subunit was positioned at the initiation codon for all variations tested. 相似文献
3.
A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3. 总被引:15,自引:3,他引:12
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The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3. 相似文献
4.
Influence of 5' proximal secondary structure on the translational efficiency of eukaryotic mRNAs and on their interaction with initiation factors 总被引:26,自引:0,他引:26
T G Lawson B K Ray J T Dodds J A Grifo R D Abramson W C Merrick D F Betsch H L Weith R E Thach 《The Journal of biological chemistry》1986,261(30):13979-13989
The effects of 5' proximal secondary structure in mRNA molecules on their translation and on their interaction with the eukaryotic initiation factors (eIF)-4F, eIF-4A, and eIF-4B have been examined. Secondary structures were generated in the 5' noncoding region of rabbit globin and reovirus mRNAs by means of hybridization with cDNA molecules. cDNAs hybridized to the first 15 bases downstream from the cap inhibited the translation of the mRNAs in both reticulocyte and wheat germ lysates. The degree of inhibition was directly related to the monovalent ion concentration and inversely related to reaction temperature. These hybrid structures also reduced the competitive ability of the messages. Hybrid structures beginning downstream from the first 15 bases did not inhibit the translation of beta-globin mRNA or reovirus s3 mRNA. None of the hybrid structures were detrimental to the interaction of the mRNAs with the 26-kDa cap binding protein of eIF-4F, as determined by chemical cross-linking assays. However, in the presence of ATP, hybrid structures immediately adjacent to the cap severely inhibited the cross-linking to the p46 subunit of eIF-4F or to additional eIF-4A or eIF-4B. In order to account for these observations, a two-step mechanism is proposed for the interaction of eIF-4F with the 5' end of an mRNA molecule. The first step involves a weak initial interaction of the p26 subunit with the cap. The second step requires the hydrolysis of ATP and results in the formation of a stable initiation factor-mRNA complex, which may involve eIF-4A and eIF-4B. This second step is inhibited by the presence of 5' proximal secondary structure. In any event, our results demonstrate that the effect of mRNA structure on translation rate depends strongly on its position with respect to the 5' end and that this effect is due at least in part to an inhibition of the action of initiation factors normally required for the unwinding of structure. 相似文献
5.
Initiation factors that bind mRNA. A comparison of mammalian factors with wheat germ factors 总被引:2,自引:0,他引:2
R D Abramson K S Browning T E Dever T G Lawson R E Thach J M Ravel W C Merrick 《The Journal of biological chemistry》1988,263(11):5462-5467
Three mammalian eukaryotic initiation factors (eIF) are required for the ATP-dependent binding of mRNA to the 40 S ribosomal subunit. These three factors, eIF-4A, eIF-4B, and eIF-4F, have also been isolated from wheat germ. Three assays were used to measure the ability of the wheat germ factors to interact with and/or substitute for the mammalian factors. Two assay systems were used to measure partial reactions involving the interaction of the three factors, ATP, and mRNA: 1) RNA-dependent ATP hydrolysis and 2) cross-linking of the factors to the 5' cap of oxidized mRNA. A third assay system was used to measure the ability of the factors to support initiation of protein synthesis. The results of the ATP hydrolysis and cross-linking experiments indicate that the wheat germ factors can interact with or substitute for the mammalian factors. Wheat germ eIF-4A appears to be functionally equivalent to mammalian eIF-4A. Wheat germ eIF-4B and eIF-4F appear to be isozymes possessing functions similar to mammalian eIF-4F. Wheat germ eIF-4B does not appear to be a functional equivalent to the mammalian eIF-4B. In a complete translation system from wheat germ, mammalian factors partially substitute for wheat germ factors, whereas the wheat germ factors are ineffective in the mammalian system. 相似文献
6.
Parsyan A Svitkin Y Shahbazian D Gkogkas C Lasko P Merrick WC Sonenberg N 《Nature reviews. Molecular cell biology》2011,12(4):235-245
The translation initiation step in eukaryotes is highly regulated and rate-limiting. During this process, the 40S ribosomal subunit is usually recruited to the 5' terminus of the mRNA. It then migrates towards the initiation codon, where it is joined by the 60S ribosomal subunit to form the 80S initiation complex. Secondary structures in the 5' untranslated region (UTR) can impede binding and movement of the 40S ribosome. The canonical eukaryotic translation initiation factor eIF4A (also known as DDX2), together with its accessory proteins eIF4B and eIF4H, is thought to act as a helicase that unwinds secondary structures in the mRNA 5' UTR. Growing evidence suggests that other helicases are also important for translation initiation and may promote the scanning processivity of the 40S subunit, synergize with eIF4A to 'melt' secondary structures or facilitate translation of a subset of mRNAs. 相似文献
7.
Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors 总被引:39,自引:0,他引:39
The assembly of initiation complexes is studied in a protein synthesis initiation assay containing ribosomal subunits, globin [125I]mRNA, [3H]Met-tRNAf, seven purified initiation factors, ATP and GTP. By omitting single components from the initiation assay, specific roles of the initiation factors, ATP and GTP are demonstrated. The initiation factor eIF-2 is required for the binding of Met-tRNAf to the 40 S ribosomal subunit. The initial Met-tRNAf binding to the small ribosomal subunit is a stringent prerequisite for the subsequent mRNA binding. The initiation factors eIF-3, eIF-4A, eIF-4B and eIF-4C together with ATP promote the binding of mRNA to the 40 S initiation complex. The association of the 40 S initiation complex with the 60 S ribosome subunit to form an 80 S initiation complex is mediated by the initiation factor eIF-5 and requires the hydrolysis of GTP. The factor eIF-1 gives a twofold overall stimulation of initiation complex formation. A model of the sequential steps in the assembly of the 80 S initiation complex in mammalian protein synthesis is presented. 相似文献
8.
Eukaryotic Translation Initiation Factor 4AIII (eIF4AIII) Is Functionally Distinct from eIF4AI and eIF4AII
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Qiyu Li Hiroaki Imataka Shigenobu Morino George W. Rogers Jr. Nancy J. Richter-Cook William C. Merrick Nahum Sonenberg 《Molecular and cellular biology》1999,19(11):7336-7346
Eukaryotic initiation factor 4A (eIF4A) is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' proximal secondary structure of eukaryotic mRNAs to facilitate attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors (eIF4E and eIF4G). Two isoforms of eIF4A, eIF4AI and eIF4AII, which are encoded by two different genes, are functionally indistinguishable. A third member of the eIF4A family, eIF4AIII, whose human homolog exhibits 65% amino acid identity to human eIF4AI, has also been cloned from Xenopus and tobacco, but its function in translation has not been characterized. In this study, human eIF4AIII was characterized biochemically. While eIF4AIII, like eIF4AI, exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, it fails to substitute for eIF4AI in an in vitro-reconstituted 40S ribosome binding assay. Instead, eIF4AIII inhibits translation in a reticulocyte lysate system. In addition, whereas eIF4AI binds independently to the middle and carboxy-terminal fragments of eIF4G, eIF4AIII binds to the middle fragment only. These functional differences between eIF4AI and eIF4AIII suggest that eIF4AIII might play an inhibitory role in translation under physiological conditions. 相似文献
9.
Ribosome binding to inosine-substituted mRNAs in the absence of ATP and mRNA factors 总被引:1,自引:0,他引:1
Incubating ribosomes and eukaryotic initiation factor eIF3 with an inosine-substituted mRNA (where the mRNA secondary structure is strongly reduced) in the absence of ATP and other protein synthesis factors produces a 40 S ribosome.mRNA complex. When Met-tRNAMeti and eIF2 are added, a 60 S ribosome subunit attaches forming an 80 S ribosome.mRNA complex. ATP and the three mRNA factors, eIF4B, cap-site factor, and eIF4A, strongly stimulate the attachment of the 60 S subunit. In the absence of Met-tRNAMeti, the 60-S subunit does not attach, and adding ATP and the mRNA factors inhibits the accumulation of 40 S ribosome.inosine mRNA complexes. These results indicate that a 40 S ribosome, probably in a complex with eIF3, has an intrinsic capacity to attach to mRNA. Further, they suggest that Met-tRNAMeti may interact in a subsequent step to stabilize the 40 S ribosome.mRNA complex and allow the attachment of a 60 S ribosome subunit. Although seen most clearly with the inosine-substituted mRNAs, the 40 S ribosome reaction is also obtained with "guanosine" mRNA. A 40 S ribosome attaches to guanosine mRNA without ATP and mRNA factors when an incubation mixture containing ribosomes, eIF3, and mRNA is fixed with glutaraldehyde. In addition, a 40 S ribosome.guanosine mRNA complex can be obtained without glutaraldehyde in incubations containing ATP and the three mRNA factors in the absence of Met-tRNAMeti. The latter reaction is limited because of the instability of the 40 S ribosome.mRNA complex in the absence of Met-tRNA. Nevertheless, its authenticity is indicated by its full dependence upon ATP and the three mRNA factors. The lack of factor requirement for the formation of 40 S ribosome complexes with inosine-substituted mRNAs indicates that ATP and the three mRNA factors function primarily to unwind the secondary structure of a guanosine mRNA. Data relevant to a role for ATP in facilitating ribosome migration on an mRNA are also discussed. 相似文献
10.
Free initiation factors eIF4A and eIF4B are dispensable for translation initiation on uncapped mRNAs
The formation of ribosomal 48S initiation complexes at the start AUG codon of uncapped mRNA leader sequences was studied using the methodology of primer extension inhibition (toe-printing). The experiments were performed in the system composed of purified individual components required for translation initiation. The formation of ribosomal 48S initiation complexes at the initiation codon was tested depending on the presence of the initiation factors eIF4F, eIF4A, and eIF4B. Several mRNAs containing short leader sequences lacking the extended secondary structure were studied. It was found that 48S ribosomal complexes at mRNAs with such leaders were not formed in the absence of eIF4F. In contrast, the removal of either eIF4A or eIF4B from the experimental system was found to be dispensable for the formation of the 48S complex. 相似文献
11.
The ATP-dependent interaction of eukaryotic initiation factors with mRNA 总被引:35,自引:0,他引:35
R D Abramson T E Dever T G Lawson B K Ray R E Thach W C Merrick 《The Journal of biological chemistry》1987,262(8):3826-3832
The interaction of three protein synthesis initiation factors, eukaryotic initiation factor (eIF)-4A, -4B, and -4F, with mRNA has been examined. Three assays specifically designed to evaluate this interaction are RNA-dependent ATP hydrolysis, retention of mRNAs on nitrocellulose filters, and cross-linking to periodate-oxidized mRNAs. The ATPase activity of eIF-4A is only activated by RNA which is lacking in secondary structure, and the minimal size of an oligonucleotide capable of effecting an optimal activation is 12-18 bases. In the presence of ATP, eIF-4A is capable of binding mRNA. Consistent with the ATPase activity, this binding shows a definite preference for single-stranded RNA. In the absence of ATP, eIF-4F is the only factor to bind capped mRNAs, and this binding, unlike that of eIF-4A, is sensitive to m7GDP inhibition. The activities of both eIF-4A and eIF-4F are stimulated by eIF-4B, which seems to have no specific independent activity in our assays. Evidence from the cross-linking studies indicates that in the absence of ATP, only the 24,000-dalton polypeptide of eIF-4F binds to the 5' cap region of the mRNA. From the data presented in conjunction with the current literature, a suggested sequence of factor binding to mRNA is: eIF-4F is the first initiation factor to bind mRNA ind an ATP-independent fashion; eIF-4B then binds to eIF-4F, if in fact it was not already bound prior to mRNA binding; and finally, eIF-4A binds to the eIF-4F X eIF-4B X mRNA complex and functions in an ATP-dependent manner to allow unwinding of the mRNA. 相似文献
12.
13.
14.
Eukaryotic translation initiation factor 4A (eIF4A) is a DEAD-box protein that participates in translation initiation. As an ATP-dependent RNA helicase, it is thought to resolve secondary structure elements from the 5′-untranslated region of mRNAs to enable ribosome scanning. The RNA-stimulated ATPase and ATP-dependent helicase activities of eIF4A are enhanced by auxiliary proteins, but the underlying mechanisms are still largely unknown. Here, we have dissected the effect of eIF4B and eIF4G on eIF4A RNA-dependent ATPase- and RNA helicase activities and on eIF4A conformation. We show for the first time that yeast eIF4B, like its mammalian counterpart, can stimulate RNA unwinding by eIF4A, although it does not affect the eIF4A conformation. The eIF4G middle domain enhances this stimulatory effect and promotes the formation of a closed eIF4A conformation in the presence of ATP and RNA. The closed state of eIF4A has been inferred but has not been observed experimentally before. eIF4B and eIF4G jointly stimulate ATP hydrolysis and RNA unwinding by eIF4A and favor the formation of the closed eIF4A conformer. Our results reveal distinct functions of eIF4B and eIF4G in synergistically stimulating the eIF4A helicase activity in the mRNA scanning process. 相似文献
15.
Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast
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Jivotovskaya AV Valásek L Hinnebusch AG Nielsen KH 《Molecular and cellular biology》2006,26(4):1355-1372
Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo. 相似文献
16.
The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity 总被引:5,自引:0,他引:5
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Shahbazian D Roux PP Mieulet V Cohen MS Raught B Taunton J Hershey JW Blenis J Pende M Sonenberg N 《The EMBO journal》2006,25(12):2781-2791
The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. Here we demonstrate that the p90 ribosomal protein S6 kinase (RSK) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3. 相似文献
17.
48S initiation complex (48S IC) formation is the first stage in the eukaryotic translation process. According to the canonical mechanism, 40S ribosomal subunit binds to the 5′-end of messenger RNA (mRNA) and scans its 5′-untranslated region (5′-UTR) to the initiation codon where it forms the 48S IC. Entire process is mediated by initiation factors. Here we show that eIF5 and eIF5B together stimulate 48S IC formation influencing initiation codon selection during ribosomal scanning. Initiation on non-optimal start codons—following structured 5′-UTRs, in bad AUG context, within few nucleotides from 5′-end of mRNA and CUG start codon—is the most affected. eIF5-induced hydrolysis of eIF2-bound GTP is essential for stimulation. GTP hydrolysis increases the probability that scanning ribosomal complexes will recognize and arrest scanning at a non-optimal initiation codon. Such 48S ICs are less stable owing to dissociation of eIF2*GDP from initiator tRNA, and eIF5B is then required to stabilize the initiator tRNA in the P site of 40S subunit. Alternative model that eIF5 and eIF5B cause 43S pre-initiation complex rearrangement favoring more efficient initiation codon recognition during ribosomal scanning is equally possible. Mutational analysis of eIF1A and eIF5B revealed distinct functions of eIF5B in 48S IC formation and subunit joining. 相似文献
18.
The eukaryotic translation factor 4A (eIF4A) is a member of DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples ATP hydrolysis to RNA binding and duplex separation. eIF4A participates in the initiation of translation by unwinding secondary structure in the 5'-untranslated region of mRNAs and facilitating scanning by the 40 S ribosomal subunit for the initiation codon. eIF4A alone has only weak ATPase and helicase activities, but these are stimulated by eIF4G, eIF4B, and eIF4H. eIF4G has two eIF4A-binding sites, one in the central domain (cp(C3)) and one in the COOH-terminal domain (cp(C2)). In the current work, we demonstrate that these two eIF4G domains have different effects on the RNA-stimulated ATPase activity of eIF4A. cp(C3) stimulates ATP-hydrolytic efficiency by about 40-fold through two mechanisms: lowering K(m)(RNA) by 10-fold and raising k(cat) by 4-fold. cp(C3) also stimulates RNA cross-linking to eIF4A in an ATP-independent manner. Studies with eIF4G and eIF4A variants suggest a model by which cp(C3) alters the conformation of the catalytic site to favor RNA binding. cp(C2) does not stimulate ATPase activity and furthermore increases both K(m)(ATP) (at saturating RNA concentrations) and K(m)(RNA) (at subsaturating ATP concentrations). Both cp(C3) and cp(C2) directly interact with the NH(2)-terminal domain of eIF4A, which possesses conserved ATP- and oligonucleotide-binding motifs, but not with the COOH-terminal domain. 相似文献
19.
A cDNA containing the complete genome of satellite tobacco necrosis virus (STNV) RNA was constructed and cloned into a plasmid vector containing the T7 polymerase promotor. A second clone containing the first 54 nucleotides from the 5' end, which includes the ribosome binding site, was also constructed. RNAs were transcribed from these plasmids (pSTNV1239 and pSTNV54) and tested for their ability to bind to wheat germ 40 S ribosomal subunits in the presence of wheat germ initiation factors eIF-4A, eIF-4F, eIF-4G, eIF-3, eIF-2, Met-tRNA, ATP, and guanosine 5'-(beta, gamma-imino)triphosphate (GMP-PNP). Maximal binding of the STNV RNA transcribed from pSTNV1239 is obtained only in the presence of all the initiation factors and ATP. In contrast, close to maximal binding of STNV RNA transcribed from pSTNV54 is obtained in the absence of eIF-4A, eIF-4F, eIF-4G, and ATP. A series of deletion clones from the 3' end of the STNV cDNA was prepared, and the requirements for binding to 40 S ribosomal subunits were determined. STNV RNAs containing more than 134 nucleotides from the 5' end require eIF-4A, eIF-4F, eIF-4G, and ATP for maximal binding to 40 S ribosomal subunits, whereas STNV RNAs containing 86 nucleotides or less no longer require ATP and these factors. These findings indicate that a region 3' to the initiation codon affects the requirements for eIF-4A, eIF-4F, eIF-4G, and ATP. 相似文献
20.
Dissociation of double-stranded polynucleotide helical structures by eukaryotic initiation factors, as revealed by a novel assay 总被引:17,自引:0,他引:17
T G Lawson K A Lee M M Maimone R D Abramson T E Dever W C Merrick R E Thach 《Biochemistry》1989,28(11):4729-4734
A new technique has been applied to the study of the RNA secondary structure unwinding activity of the eukaryotic initiation factors (eIFs) 4F, 4A, and 4B. Secondary structures were generated at the 5' ends of reovirus and globin mRNA molecules by hybridization with 32P-labeled cDNA molecules 15 nucleotide residues long. The dissociation of the labeled cDNAs from the mRNAs was assayed by a gel filtration chromatography procedure which separates the free cDNAs from mRNAs and mRNA/cDNA hybrids. When the three factors were tested alone, only eIF-4F stimulated dissociation of hybrids. The combination of eIF-4A plus eIF-4B also exhibited a strong hybrid dissociating activity, which was markedly temperature dependent. Under optimum conditions, up to 90% of the hybrid structures are disrupted in 60 min. These results demonstrate for the first time that stable double-stranded regions can be melted and dissociated by eIFs. They also characterize more precisely the first step in the structure unwinding reaction. 相似文献