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1.
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
2.
K Balkow S Mizuno M Rabinovitz 《Biochemical and biophysical research communications》1973,54(1):315-323
The trinucleotide codon, AUG, promotes the combination of reticulocyte 40S and 60S native ribosomal subunits as well as recombination of those derived by dissociation factor. This interaction is inhibited by hemin deficiency or the hemin-controlled repressor and results in the loss of methionine from ribosomal bound met-tRNAf. The locus of inhibition among the partial reactions of peptide chain initiation is between met-tRNAf and initiation codon binding to the 40S ribosomal subunit and peptide bond formation. 相似文献
3.
S. U. Åström M. E. Nordlund F. L. Erickson E. M. Hannig A. S. Byström 《Molecular genetics and genomics : MGG》1999,261(6):967-976
The Saccharomyces cerevisiae gene RIT1 encodes a phospho-ribosyl transferase that exclusively modifies the initiator tRNA (tRNAMet i) by the addition of a 2′-O-ribosyl phosphate group to Adenosine 64. As a result, tRNAMet i is prevented from participating in the elongation steps of protein synthesis. We previously showed that the modification is not essential for the function of tRNAMet i in the initiation of translation, since rit1 null strains are viable and show no obvious growth defects. Here, we demonstrate that yeast strains in which a rit1 null allele is combined with mutations in any of the genes for the three subunits of eukaryotic initiation factor-2 (eIF-2), or with disruption alleles of two of the four initiator methionine tRNA (IMT) genes, show synergistic growth defects. A multicopy plasmid carrying an IMT gene can alleviate these defects. On the other hand, introduction of a high-copy-number plasmid carrying the TEF2 gene, which encodes the eukaryotic elongation factor 1α (eEF-1α), into rit1 null strains with two intact IMT genes had the opposite effect, indicating that increased levels of eEF-1α are deleterious to these strains, presumably due to sequestration of the unmodified met-tRNAMet i for elongation. Thus, under conditions in which the components of the ternary met-tRNAMet i:GTP:eIF-2 complex become limiting or are functionally impaired, the presence of the 2′-O-ribosyl phosphate modification in tRNAMet i is important for the provision of adequate amounts of tRNAMet i for formation of this ternary complex. 相似文献
4.
S. U. ?str?m M. E. Nordlund F. L. Erickson E. M. Hannig A. S. Bystr?m 《Molecular & general genetics : MGG》1999,261(6):967-976
The Saccharomyces cerevisiae gene RIT1 encodes a phospho-ribosyl transferase that exclusively modifies the initiator tRNA (tRNAMet
i) by the addition of a 2′-O-ribosyl phosphate group to Adenosine 64. As a result, tRNAMet
i is prevented from participating in the elongation steps of protein synthesis. We previously showed that the modification
is not essential for the function of tRNAMet
i in the initiation of translation, since rit1 null strains are viable and show no obvious growth defects. Here, we demonstrate that yeast strains in which a rit1 null allele is combined with mutations in any of the genes for the three subunits of eukaryotic initiation factor-2 (eIF-2),
or with disruption alleles of two of the four initiator methionine tRNA (IMT) genes, show synergistic growth defects. A multicopy plasmid carrying an IMT gene can alleviate these defects. On the other hand, introduction of a high-copy-number plasmid carrying the TEF2 gene, which encodes the eukaryotic elongation factor 1α (eEF-1α), into rit1 null strains with two intact IMT genes had the opposite effect, indicating that increased levels of eEF-1α are deleterious to these strains, presumably due
to sequestration of the unmodified met-tRNAMet
i for elongation. Thus, under conditions in which the components of the ternary met-tRNAMet
i:GTP:eIF-2 complex become limiting or are functionally impaired, the presence of the 2′-O-ribosyl phosphate modification in
tRNAMet
i is important for the provision of adequate amounts of tRNAMet
i for formation of this ternary complex.
Received: 20 November 1998 / Accepted: 7 April 1999 相似文献
5.
Steve Legon Anne Brayley Tim Hunt Richard J. Jackson 《Biochemical and biophysical research communications》1974,56(3):745-752
Cyclic AMP and a variety of purines are able to overcome the inhibition of the initiation of protein synthesis caused by incubation of the lysate in the absence of added hemin or with double-stranded RNA or oxidised glutathione. These three inhibitions show similar kinetics and are each accompanied by the disappearance of a complex between the 40S ribosomal subunits and met-tRNAf. A translational repressor has been implicated in the inhibition seen in the absence of hemin and we suggest that the link between these three inhibitions is the accumulation of this repressor. 相似文献
6.
Lang CH Frost RA 《American journal of physiology. Endocrinology and metabolism》2004,287(4):E721-E730
Polymicrobial sepsis impairs skeletal muscle protein synthesis, which results from impairment in translation initiation under basal conditions. The purpose of the present study was to test the hypothesis that sepsis also impairs the anabolic response to amino acids, specifically leucine (Leu). Sepsis was induced by cecal ligation and puncture, and 24 h later, Leu or saline (Sal) was orally administered to septic and time-matched nonseptic rats. The gastrocnemius was removed 20 min later for assessment of protein synthesis and signaling components important in peptide-chain initiation. Oral Leu increased muscle protein synthesis in nonseptic rats. Leu was unable to increase protein synthesis in muscle from septic rats, and synthetic rates remained below those observed in nonseptic + Sal rats. In nonseptic + Leu rats, phosphorylation of eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1) in muscle was markedly increased compared with values from time-matched Sal-treated nonseptic rats. This change was associated with redistribution of eIF4E from the inactive eIF4E.4E-BP1 to the active eIF4E.eIF4G complex. In septic rats, Leu-induced phosphorylation of 4E-BP1 and changes in eIF4E distribution were completely abrogated. Sepsis also antagonized the Leu-induced increase in phosphorylation of S6 kinase 1 and ribosomal protein S6. Sepsis attenuated Leu-induced phosphorylation of mammalian target of rapamycin and eIF4G. The ability of sepsis to inhibit anabolic effects of Leu could not be attributed to differences in plasma concentrations of insulin, insulin-like growth factor I, or Leu between groups. In contrast, the ability of exogenous insulin-like growth factor I to stimulate the same signaling components pertaining to translation initiation was not impaired by sepsis. Hence, sepsis produces a relatively specific Leu resistance in skeletal muscle that impairs the ability of this amino acid to stimulate translation initiation and protein synthesis. 相似文献
7.
Bernhard Erni Theophil Staehelin 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,740(4):373-378
Binding of the Met-tRNAMetf·eIF-2 GTP complex to the 40 S ribosomal subunit is the first step in initiation of eukaryotic protein synthesis. The extent of binding and the stability of the complex are enhanced by initiation factors eIF-3 and eIF-4C, AUG and elevated magnesium concentration. The reversibility of reaction steps occurring during the assembly of the initiation complex is measured as the rate of Met-tRNAMetf exchange in the initiation complex and its intermediates. This rate progressively decreases and Met-tRNAMetf binding becomes irreversible upon binding of mRNA. The association of the 40 S Met-tRNAMetf mRNA initiation complex with the 60 S ribosomal subunit is again reversible as long as elongation does not occur. 相似文献
8.
James S. Hutchison Kivie Moldave 《Biochemical and biophysical research communications》1981,99(2):722-728
The effect of elevated temperature on the activity of various components involved in protein synthesis was investigated in extracts from cultured Chinese hamster ovary cells. The translation of exogenous mRNA was markedly inhibited by preincubation of the extract for 15 to 20 minutes at 42°C. However, the following intermediary reactions were not affected, or only slightly inhibited, at 42°C: 1) the incorporation of Met-tRNAf into eIF-2·Met-tRNAf·GTP ternary complex; 2) the interaction of the ternary complex with 40S ribosomal subunits to form the 40S preinitiation intermediate; 3) the binding of mRNA and 60S subunits to form the 80S initiation complex; and 4) the reactions catalyzed by elongation factors EF-1 and EF-2. The activity of Met-tRNA synthetase was markedly inhibited, affecting the formation of initiator Met-tRNAf required for the initiation of protein synthesis and the translation of natural mRNA. Other aminoacyl-tRNA synthetases were not significantly affected by the elevated temperature. 相似文献
9.
Initiation of protein synthesis: evidence for messenger RNA-independent binding of methionyl-transfer RNA to the 40 S ribosomal subunit 总被引:45,自引:0,他引:45
This paper shows that reticuloeyte lysates contain 40 S/Met-tRNAf complexes which are intermediates in the initiation of protein synthesis before the involvement of messenger RNA. More than one third of the native 40 S subunits in the lysate exist as these complexes during periods of linear protein synthesis, but less than a tenth are associated with mRNA.The 40 S/Met-tRNAf complexes disappear in some situations in which initiation is inhibited (by double-stranded RNA, oxidized glutathione, or in the absence of added haemin), but persist in the presence of other inhibitors (e.g. aurintricarboxylate or poly(I)). Inhibitors of chain elongation had little effect on the amount of these complexes.The Met-tRNAf in the 40 S complexes appears to exchange readily with free Met-tRNAf; when lysates were preincubated with sparsomycin or diphtheria toxin and then incubated with [35S]Met-tRNAf, the native 40 S subunits were the only ribosomal particles labelled. This experimental system was used to examine whether 40 S/Met-tRNAf complexes could interact with mRNA; various mRNAs were added shortly after or at the same time as the [35S]Met-tRNAf. This resulted in a conversion of the 40 S/Met-tRNAf complexes into 80 S complexes, which appeared to be true initiation complexes since they were capable of translating the first two codons of the added mRNA. The mRNA-dependent formation of these 80 S complexes was completely inhibited by 0.1 mM-aurintricarboxylate, but the association of Met-tRNAf with the 40 S subunits was not prevented.The 40 S/Met-tRNAf complexes also participated in initiation on endogenous mRNA, and it was shown that the Met-tRNAf in this complex was used in preference to free Met-tRNAf in this process.We propose that the first step in the initiation of protein synthesis in the reticuloeyte lysate is the formation of a 40 S/Met-tRNAf complex. In the second stage the complex binds mRNA at the correct initiation site and, after joining with a 60 S subunit, an 80 S/Met-tRNAf/mRNA initiation complex is formed. 相似文献
10.
Renger G 《Photosynthesis research》2007,92(3):407-425
This minireview is an attempt to summarize our current knowledge on oxidative water splitting in photosynthesis. Based on
the extended Kok model (Kok, Forbush, McGloin (1970) Photochem Photobiol 11:457–476) as a framework, the energetics and kinetics
of two different types of reactions comprising the overall process are discussed: (i) P680+• reduction by the redox active tyrosine YZ of polypeptide D1 and (ii) Yzox induced oxidation of the four step sequence in the water oxidizing complex (WOC) leading to the formation of molecular oxygen.
The mode of coupling between electron transport (ET) and proton transfer (PT) is of key mechanistic relevance for the redox
turnover of YZ and the reactions within the WOC. The peculiar energetics of the oxidation steps in the WOC assure that redox state S1 is thermodynamically most stable. This is a general feature in all oxygen evolving photosynthetic organisms and assumed to
be of physiological relevance. The reaction coordinate of oxidative water splitting is discussed on the basis of the available
information about the Gibbs energy differences between the individual redox states S
i+1 and S
i
and the data reported for the activation energies of the individual oxidation steps in the WOC. Finally, an attempt is made
to cast our current state of knowledge into a mechanism of oxidative water splitting with special emphasis on the formation
of the essential O–O bond and on the active role of the protein in tuning the local proton activity that depends on time and
redox state S
i
. The O–O linkage is assumed to take place at the level of a complexed peroxide. 相似文献
11.
Pilar Martin-Marcos Jagpreet S. Nanda Rafael E. Luna Fan Zhang Adesh K. Saini Vera A. Cherkasova Gerhard Wagner Jon R. Lorsch Alan G. Hinnebusch 《RNA (New York, N.Y.)》2014,20(2):150-167
In the current model of translation initiation by the scanning mechanism, eIF1 promotes an open conformation of the 40S subunit competent for rapidly loading the eIF2·GTP·Met-tRNAi ternary complex (TC) in a metastable conformation (POUT) capable of sampling triplets entering the P site while blocking accommodation of Met-tRNAi in the PIN state and preventing completion of GTP hydrolysis (Pi release) by the TC. All of these functions should be reversed by eIF1 dissociation from the preinitiation complex (PIC) on AUG recognition. We tested this model by selecting eIF1 Ssu− mutations that suppress the elevated UUG initiation and reduced rate of TC loading in vivo conferred by an eIF1 (Sui−) substitution that eliminates a direct contact of eIF1 with the 40S subunit. Importantly, several Ssu− substitutions increase eIF1 affinity for 40S subunits in vitro, and the strongest-binding variant (D61G), predicted to eliminate ionic repulsion with 18S rRNA, both reduces the rate of eIF1 dissociation and destabilizes the PIN state of TC binding in reconstituted PICs harboring Sui− variants of eIF5 or eIF2. These findings establish that eIF1 dissociation from the 40S subunit is required for the PIN mode of TC binding and AUG recognition and that increasing eIF1 affinity for the 40S subunit increases initiation accuracy in vivo. Our results further demonstrate that the GTPase-activating protein eIF5 and β-subunit of eIF2 promote accuracy by controlling eIF1 dissociation and the stability of TC binding to the PIC, beyond their roles in regulating GTP hydrolysis by eIF2. 相似文献
12.
Kir1.1 channels are important in maintaining K+ homeostasis in the kidney. Intracellular acidification reversibly closes the Kir1.1 channel and thus decreases K+ secretion. In this study, we used Foster resonance energy transfer (FRET) to determine whether the conformation of the cytoplasmic
pore changes in response to intracellular pH (pHi)-gating in Kir1.1 channels fused with enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP)
(ECFP-Kir1.1-EYFP). Because the fluorescence intensities of ECFP and EYFP were affected at pHi < 7.4 where pHi-gating occurs in the ECFP-Kir1.1-EYFP construct, we examined the FRET efficiencies of an ECFP-S219R-EYFP mutant, which is
completed closed at pHi 7.4 and open at pHi 10.0. FRET efficiency was increased from 25% to 40% when the pHi was decreased from 10.0 to 7.4. These results suggest that the conformation of the cytoplasmic pore in the Kir1.1 channel
changes in response to pHi gating such that the N- and C-termini move apart from each other at pHi 7.4, when the channel is open. 相似文献
13.
Adesh K. Saini Jagpreet S. Nanda Pilar Martin-Marcos Jinsheng Dong Fan Zhang Monika Bhardwaj Jon R. Lorsch Alan G. Hinnebusch 《Nucleic acids research》2014,42(15):9623-9640
eIF5 is the GTPase activating protein (GAP) for the eIF2·GTP·Met-tRNAiMet ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which Pi is released from eIF2·GDP·Pi. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and Pi release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui− mutations in numerous factors. We conclude that both of eIF5''s functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in
vivo. 相似文献
14.
A cell-free system from cultured Chinese hamster ovary cells has been developed, which translates endogenous mRNAs, exogenous natural mRNAs, and synthetic polynucleotide templates. The analysis of most of the reactions involved in initiation, elongation, and termination of protein synthesis can be carried out in this system. The postmitochondrial fraction, containing ribosomal 40 and 60 S subunits, 80 S ribosomes, polysomes, and cytosol proteins, incorporates amino acids into protein. The preparation is capable of recycling endogenous mRNA by initiating protein synthesis on polysomal mRNA, and of initiating protein synthesis on exogenous templates. When endogenous mRNA is degraded with micrococcal nuclease, polysomes are no longer evident and protein synthesis is markedly depended on added mRNA, ATP, GTP, and a nucleoside triphosphate-generating system. Amino acid incorporation is linear for over 2 h, polysomes containing nascent polypeptide chains are reformed and, with time, most of the protein synthesized is released into the media. Gel electrophoretic analysis of the product formed in response to globin mRNA indicates that most of the radioactivity migrates as a single peak, in the region corresponding to globin. Comparison of the electrophoretic pattern obtained from labeled Chinese hamster ovary cells with that from incubations of cell extract and Chinese hamster ovary mRNA indicates that essentially all of the polypeptides formed by the intact cell are synthesized by the cell-free system. Sucrose gradient centrifugation of incubations containing mRNA-depleted extract and [35S]methionine, in the absence of added mRNA, is used to detect initiation intermediates in the formation of the [40 S Met-tRNAf] complex and, with added natural mRNA plus cycloheximide, to detect intermediates in the formation of the 80 S initiation complex. Chain elongation reactions are measured by the incorporation of [3H]phenylalanine into polyphenylalanine in extracts supplemented with poly(U), or by the formation of nascent polypeptide chains on polysomes with natural mRNA. Chain termination is measured by analyzing the amount of radioactive protein released into the cytosol. 相似文献
15.
Mark Stephen Kirby Clive Orchard Mark Richard Boyett 《Molecular and cellular biochemistry》1989,89(2):109-113
The role of Ca2+ in the initiation and maintenance of contraction has been extensively studies. Many of these studies have focused on how Ca2+ influx and efflux affect cytoplasmic Ca2+ (Cai) and, therefore, contraction in cardiac muscle. However, it has recently become apparent that Cai itself may play a major role in the control of Ca2+ influx and efflux from cardiac muscle. Here we review current ideas on the mechanisms underlying Ca2+ homeostasis in cardiac muscle, with specific attention to how Cai may control Ca2+ influx, both under normal and pathological conditions. 相似文献
16.
《生物化学与生物物理学报:生物膜》1985,819(2):267-270
Activation of Na+-H+ exchange in rat thymocytes was found to be followed by an increase in free cytoplasmic Ca2+ concentration ([Ca2+]i). We determined whether the change in [Ca2+]i was secondary to the uptake of Na+, or to the cytoplasmic alkalinization that result from activation of the antiport. Increasing intracellular [Na+] by treating the cells with ouabain or gramicidin failed to affect [Ca2+]i. In contrast, procedures that increased the cytoplasmic pH, such as addition of monensin or NH3, significantly elevated [Ca2+]i. These results suggest an important role of cytoplasmic pH in the control of [Ca2+]i in lymphocytes. 相似文献
17.
18.
Arnab Ghosh Supriya Jindal Amber A. Bentley Alan G. Hinnebusch Anton A. Komar 《Nucleic acids research》2014,42(13):8537-8555
Conserved ribosomal proteins frequently harbor additional segments in eukaryotes not found in bacteria, which could facilitate eukaryotic-specific reactions in the initiation phase of protein synthesis. Here we provide evidence showing that truncation of the N-terminal domain (NTD) of yeast Rps5 (absent in bacterial ortholog S7) impairs translation initiation, cell growth and induction of GCN4 mRNA translation in a manner suggesting incomplete assembly of 48S preinitiation complexes (PICs) at upstream AUG codons in GCN4 mRNA. Rps5 mutations evoke accumulation of factors on native 40S subunits normally released on conversion of 48S PICs to 80S initiation complexes (ICs) and this abnormality and related phenotypes are mitigated by the SUI5 variant of eIF5. Remarkably, similar effects are observed by substitution of Lys45 in the Rps5-NTD, involved in contact with Rps16, and by eliminating the last two residues of the C-terminal tail (CTT) of Rps16, believed to contact initiator tRNA base-paired to AUG in the P site. We propose that Rps5-NTD-Rps16-NTD interaction modulates Rps16-CTT association with Met-tRNAiMet to promote a functional 48S PIC. 相似文献
19.
PROTEIN synthesis in at least some mammalian cells is probably initiated by Met-tRNAf1–3, which binds to salt-washed ribosomes at low Mg2+ concentrations in the presence of AUG and initiation factors4,5. Myosin mRNA will bind to 40S ribosomal subunits and if this represents a true initiation complex, it should bind specific initiator tRNA6,7. We report that an initiation complex specific for Met-tRNAf can be formed with the 40S ribosomal subunit isolated from mouse plasmacytoma tumours. 相似文献
20.
Taehee Kang Nam Hoon Kwon Jin Young LeeMin Chul Park Eunji KangHyo Hyun Kim Taek Jin KangSunghoon Kim 《Journal of molecular biology》2012,423(4):475-481
Aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMPs) are nonenzymatic scaffolding proteins that comprise multisynthetase complex (MSC) with nine aminoacyl-tRNA synthetases in higher eukaryotes. Among the three AIMPs, AIMP3/p18 is strongly anchored to methionyl-tRNA synthetase (MRS) in the MSC. MRS attaches methionine (Met) to initiator tRNA (tRNAiMet) and plays an important role in translation initiation. It is known that AIMP3 is dispatched to nucleus or nuclear membrane to induce DNA damage response or senescence; however, the role of AIMP3 in translation as a component of MSC and the meaning of its interaction with MRS are still unclear. Herein, we observed that AIMP3 specifically interacted with Met-tRNAiMetin vitro, while it showed little or reduced interaction with unacylated or lysine-charged tRNAiMet. In addition, AIMP3 discriminates Met-tRNAiMet from Met-charged elongator tRNA based on filter-binding assay. Pull‐down assay revealed that AIMP3 and MRS had noncompetitive interaction with eukaryotic initiation factor 2 (eIF2) γ subunit (eIF2γ), which is in charge of binding with Met-tRNAiMet for the delivery of Met-tRNAiMet to ribosome. AIMP3 recruited active eIF2γ to the MRS-AIMP3 complex, and the level of Met-tRNAiMet bound to eIF2 complex was reduced by AIMP3 knockdown resulting in reduced protein synthesis. All these results suggested the novel function of AIMP3 as a critical mediator of Met-tRNAiMet transfer from MRS to eIF2 complex for the accurate and efficient translation initiation. 相似文献