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1.
《Critical reviews in biochemistry and molecular biology》2013,48(4-5):385-402
AbstractAlthough recent biochemical and genetic investigations have produced some insights into the mechanism of initiation of translation in eukaryotic cells, two aspects of the initiation process remain controversial. One unsettled issue concerns a variety of functions that have been proposed for mRNA binding proteins, including some initiation factors. The need to distinguish between specific and nonspecific binding of proteins to mRNA is discussed herein. The possibility that certain initiation factors might act as RNA helicases is evaluated along with other ideas about the functions of mRNA- and ATP-binding factors. A second controversial issue concerns the universality of the scanning mechanism for initiation of translation. According to the conventional scanning model, the initial contact between eukaryotic ribosomes and mRNA occurs exclusively at the 5' terminus of the message, which is usually capped. The existence of uncapped mRNAs among a few plant and animal viruses has prompted a vigorous search for other modes of initiation. An “internal initiation” mechanism, first proposed for picornaviruses, has received considerable attention. Although a large body of evidence has been adduced in support of such a mechanism, many of the experiments appear flawed or inconclusive. Some suggestions are given for improving experiments designed to test the internal initiation hypothesis. 相似文献
2.
Initiation of translation in prokaryotes and eukaryotes. 总被引:74,自引:0,他引:74
3.
Tokumasa Nakamoto 《Molecular biology reports》2011,38(2):847-855
The various mechanisms proposed to describe the initiation of protein synthesis are reviewed with a focus on their initiation
signals. A characteristic feature of the various mechanisms is that each one of them postulates a distinct initiation signal.
The signals of the Shine–Dalgarno (SD), the scanning and the internal ribosome entry site (IRES) mechanisms are all located
exclusively in the 5′ leader sequence, whereas, the signal of the cumulative specificity (CS) mechanism includes the entire
initiation site (IS). Computer analysis of known E. coli IS sequences showed signal characteristics in the entire model IS consisting of 47 bases, in segments of the 5′ leader and
of the protein-coding regions. The proposal that eukaryotic translation actually occurs in two steps is scrutinized. In a
first step, initiation factors (eIF4F) interact with the cap of the mRNA, thereby enhancing the accessibility of the IS. In
the second step, initiation is by the conserved prokaryotic mechanism in which the ribosomes bind directly to the mRNA without
ribosomal scanning. This binding occurs by the proposed process of in reading frame binding of ribosomes to mRNA, which is
consistent with the CS mechanism. The basic CS mechanism is able to account for the initiation of translation of leaderless
mRNAs, as well as for that of canonical mRNAs. The SD, the scanning and the IRES mechanisms, on the other hand, are inconsistent
with the initiation of translation of leaderless mRNAs. Based on these and other observations, it is deemed that the CS mechanism
is the universal initiation mechanism. 相似文献
4.
Butland G Krogan NJ Xu J Yang WH Aoki H Li JS Krogan N Menendez J Cagney G Kiani GC Jessulat MG Datta N Ivanov I Abouhaidar MG Emili A Greenblatt J Ganoza MC Golshani A 《Journal of cellular biochemistry》2007,100(3):642-652
Here, we report the use of an in vivo protein-protein interaction detection approach together with focused follow-up experiments to study the function of the DeaD protein in Escherichia coli. In this method, functions are assigned to proteins based on the interactions they make with others in the living cell. The assigned functions are further confirmed using follow-up experiments. The DeaD protein has been characterized in vitro as a putative prokaryotic factor required for the formation of translation initiation complexes on structured mRNAs. Although the RNA helicase activity of DeaD has been demonstrated in vitro, its in vivo activity remains controversial. Here, using a method called sequential peptide affinity (SPA) tagging, we show that DeaD interacts with certain ribosomal proteins as well as a series of other nucleic acid binding proteins. Focused follow-up experiments provide evidence for the mRNA helicase activity of the DeaD protein complex during translation initiation. DeaD overexpression compensates for the reduction of the translation activity caused by a structure placed at the initiation region of a chloramphenicol acetyltransferase gene (cat) used as a reporter. Deletion of the deaD gene, encoding DeaD, abolishes the translation activity of the mRNA with an inhibitory structure at its initiation region. Increasing the growth temperature disrupts RNA secondary structures and bypasses the DeaD requirement. These observations suggest that DeaD is involved in destabilizing mRNA structures during translation initiation. This study also provides further confirmation that large-scale protein-protein interaction data can be suitable to study protein functions in E. coli. 相似文献
5.
6.
It is generally accepted that the initiation of translation in eukaryotes involves the binding of the 40S ribosomal subunit to the capped 5′ end of an mRNA and subsequent scanning of 5′ UTR in search of an initiation codon. However, until recently this has remained a mere hypothesis. This review describes the novel experimental evidence in support of this classical model. Data on the participation of various factors in the eukaryotic initiation process are summarized. The sequence of initiation events is described in light of the latest experimental data. The existing physical models of scanning are presented. Special attention is paid to discussion of alternative models of eukaryotic initiation of translation. It is demonstrated that the canonical mechanism of initiation is more versatile than previously thought. 相似文献
7.
The mechanism by which internal ribosomal binding on the picornaviral RNA takes place is still not known. An important role has been suggested for eukaryotic initiation factors eIF-4A, eIF-4B, as well as for some not yet defined trans-acting factors like p52 for poliovirus and p58 for encephalomyocarditis virus (EMCV). In this paper we describe the competition between the 5' untranslated region (UTR) of EMCV and globin mRNA for the translational apparatus in rabbit reticulocyte lysates and show that the factor that is competed for is eIF-2/2B. The EMC 5' UTR is a very strong inhibitor of globin synthesis in the rabbit reticulocyte lysate because of a 30-fold higher eIF-2/2B binding capacity. Mutations 100 to 140 nucleotides upstream of the initiation codon led to a decreased efficiency to initiate translation and to a decreased ability to inhibit globin mRNA translation. The results suggest an important role for eIF-2/2B binding in EMC RNA translation and therefore in internal initiation. 相似文献
8.
The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation. 总被引:9,自引:0,他引:9
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Translation initiation on poliovirus and encephalomyocarditis virus (EMCV) mRNAs occurs by a cap-independent mechanism utilizing an internal ribosomal entry site (IRES). However, no unifying mechanism for AUG initiation site selection has been proposed. Analysis of initiation of mRNAs translated in vitro has suggested that initiation of poliovirus mRNA translation likely involves both internal binding of ribosomes and scanning to the first AUG which is in a favorable context for initiation. In contrast, internal initiation on EMCV mRNA may not utilize scanning, since ribosomes bind directly or very close to the initiation codon AUG-11. We have studied in vivo the sequence requirements for internal initiation around the EMCV initiation codon, both in monocistronic and in dicistronic mRNAs. Our studies show that the upstream AUG-10 is normally not used and that there is no specific sequence requirement for nucleotides between AUG-10 and AUG-11. However, the sequence context of AUG-11 does influence the efficiency of initiation at AUG-11. Efficient IRES-mediated internal initiation at AUG-11 exhibits a requirement for an adenine in the -3 position, similar to cap-dependent initiation. These results support a model for internal initiation on EMCV mRNA in which scanning starts at or near AUG-11. Although initiation primarily occurs at AUG-11, initiation at multiple downstream AUG codons can be detected. In addition, a poor sequence context around AUG-11 results in increased initiation at one or more downstream AUG codons, indicative of leaky scanning or jumping by the ribosome from AUG-11 mediated by the EMCV IRES. 相似文献
9.
Translation of poliovirus mRNA 总被引:5,自引:0,他引:5
Ribosome binding to cellular eukaryotic mRNAs is proposed to occur by initial attachment at or near the mRNA 5' cap structure (m7 GppN, where N is any nucleotide) followed by scanning till the appropriate initiator AUG is encountered. A pivotal aspect of this model is the obligatory entry of the ribosomes at the 5' end of the mRNA (regardless if it contains a cap structure). Recent experiments, however, demonstrated that ribosomes can access certain mRNAs by internal binding to the 5'-untranslated region. This was most clearly demonstrated for members of the picornavirus family such as poliovirus and encephalomyocarditis virus. Further experiments suggest that other viral mRNAs and even cellular mRNAs may use similar mechanisms of ribosome binding. Here we describe some features of the poliovirus 5'-untranslated region and possible trans-acting factors that are involved in this mechanism of translation. 相似文献
10.
The involvement of mRNA secondary structure in protein synthesis 总被引:15,自引:0,他引:15
Translation initiation in eukaryotes is a complex process involving many factors. A key step in this process is the binding of mRNA to the 43S preinitiation complex. This is generally the rate-limiting step in translation initiation and consequently a major determinant of mRNA translational efficiency. The primary and secondary structure of the mRNA 5' noncoding region have been implicated in modulating translational efficiency. Translational efficiency was shown to be inversely proportional to the degree of secondary structure at the mRNA 5' noncoding region. Furthermore, it was shown that cap-binding proteins that interact with the 5' cap structure (m7GpppN) of eukaryotic mRNAs are involved in the "unwinding" of the mRNA secondary structure, in an ATP hydrolysis mediated event, to facilitate ribosome binding. Thus, cap-binding proteins can potentially regulate mRNA translation. Here, we discuss the available data supporting the notion that eukaryotic 5' mRNA secondary structure plays an important role in translation initiation and the possible regulation of this process. 相似文献
11.
S. E. Dmitriev D. E. Andreev Z. V. Adyanova I. M. Terenin I. N. Shatsky 《Molecular Biology》2009,43(1):108-113
According to the generally accepted scanning model proposed by M. Kozak, the secondary structure of the 5′-untranslated region (5′-UTR) of eukaryotic mRNA can only inhibit the translation initiation by counteracting migration of the 40S ribosomal subunit along the mRNA polynucleotide chain. The existence of efficiently translatable mRNAs with long and highly structured 5′-UTRs is incompatible with the cap-dependent scanning mechanism. Such mRNAs are expected to use alternative ways of translation initiation to be efficiently translated, primarily the mechanism of internal ribosome entry mediated by internal ribosome entry sites (IRESs), special RNA structures that reside in the 5′-UTR. The paper shows that this viewpoint is incorrect and is probably based on experiments with mRNA translation in rabbit reticulocyte lysate. This cell-free system fails to adequately reflect the relative translation efficiencies observed for different mRNAs in vivo. Five structurally similar mRNAs with either short leaders of the β-globin and β-actin mRNAs or long and highly structured 5′-UTRs of the c-myc, LINE-1, and Apaf-1 mRNAs displayed comparable translation activities in transfected cells and an entire cytoplasmic extract of cultivated cells. Translation activity proved to strongly depend on the presence of a cap at the 5′ end. 相似文献
12.
Matías Blaustein Leandro Quadrana Guillermo Risso Manuel de la Mata Federico Pelisch Anabella Srebrow 《Journal of cellular biochemistry》2009,107(4):826-833
Post‐splicing activities have been described for a subset of shuttling serine/arginine‐rich splicing regulatory proteins, among them SF2/ASF. We showed that growth factors activate a Ras‐PI 3‐kinase‐Akt/PKB signaling pathway that not only modifies alternative splicing of the fibronectin EDA exon, but also alters in vivo translation of reporter mRNAs containing the EDA binding motif for SF2/ASF, providing two co‐regulated levels of isoform‐specific amplification. Translation of most eukaryotic mRNAs is initiated via the scanning mechanism, which implicates recognition of the m7G cap at the mRNA 5′‐terminus by the eIF4F protein complex. Several viral and cellular mRNAs are translated in a cap‐independent manner by the action of cis‐acting mRNA elements named internal ribosome entry sites that direct internal ribosome binding to the mRNA. Here we use bicistronic reporters that generate mRNAs carrying two open reading frames, one translated in a cap‐dependent manner while the other by internal ribosome entry site‐dependent initiation, to show that in vivo over‐expression of SF2/ASF increases the ratio between cap‐dependent and internal ribosome entry site‐dependent translation. Consistently, knocking‐down of SF2/ASF causes the opposite effect. Changes in expression levels of SF2/ASF also affect alternative translation of an endogenous mRNA, that one coding for fibroblast growth factor‐2. These results strongly suggest a role for SF2/ASF as a regulator of alternative translation, meaning the generation of different proteins by the balance among these two translation initiation mechanisms, and expand the known potential of SF2/ASF to regulate proteomic diversity to the translation field. J. Cell. Biochem. 107: 826–833, 2009. © 2009 Wiley‐Liss, Inc. 相似文献
13.
Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation. 总被引:32,自引:5,他引:27
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Eukaryotic translation initiation factor-4A (eIF-4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA-dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA-ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF-4F. To gain further understanding into the mechanism of action of eIF-4A in mRNA-ribosome binding, defective eIF-4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF-4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap-independent internal initiation mechanism. Addition of eIF-4A or eIF-4F relieved inhibition of translation, but eIF-4F was six times more effective than eIF-4A, whereas eIF-4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF-4A is defective in recycling through eIF-4F, thus explaining the dramatic inhibition of translation. Mutant eIF-4A proteins also inhibited eIF-4F-dependent, but not eIF-4A-dependent RNA helicase activity. Taken together these results suggest that eIF-4A functions primarily as a subunit of eIF-4F, and that singular eIF-4A is required to recycle through the complex during translation. Surprisingly, eIF-4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs. 相似文献
14.
15.
During the last decade the concept of cellular IRES-elements has become predominant to explain the continued expression of
specific proteins in eukaryotic cells under conditions when the cap-dependent translation initiation is inhibited. However,
many cellular IRESs regarded as cornerstones of the concept, have been compromised by several recent works using a number
of modern techniques. This review analyzes the sources of artifacts associated with identification of IRESs and describes
a set of control experiments, which should be performed before concluding that a 5’ UTR of eukaryotic mRNA does contain an
IRES. Hallmarks of true IRES-elements as exemplified by well-documented IRESs of viral origin are presented. Analysis of existing
reports allows us to conclude that there is a constant confusion of the cap-independent with the IRES-directed translation
initiation. In fact, these two modes of translation initiation are not synonymous. We discuss here not numerous reports pointing
to the existence of a cap- and IRES-independent scanning mechanism of translation initiation based on utilization of special
RNA structures called cap-independent translational enhancers (CITE). We describe this mechanism and suggest it as an alternative
to the concept of cellular IRESs. 相似文献
16.
In response to environmental stress and viral infection, mammalian cells form foci containing translationally silenced mRNPs termed stress granules (SGs). As aggregates of stalled initiation complexes, SGs are defined by the presence of translation initiation machinery in addition to mRNA binding proteins. Here, we report that cells infected with poliovirus (PV) can form SGs early that contain T-cell-restricted intracellular antigen 1 (TIA1), translation initiation factors, RNA binding proteins, and mRNA. However, this response is blocked as infection progresses, and a type of pseudo-stress granule remains at late times postinfection and contains TIA but lacks translation initiation factors, mRNA binding proteins, and most polyadenylated mRNA. This result was observed using multiple stressors, including viral infection, oxidative stress, heat shock, and endoplasmic reticulum stress. Multiple proteins required for efficient viral internal ribosome entry site-dependent translation are localized to SGs under stress conditions, providing a potential rationale for the evolution and maintenance of the SG inhibition phenotype. Further, the expression of a noncleavable form of the RasGAP-SH3 domain binding protein in PV-infected cells enables SGs whose constituents are consistent with the presence of stalled 48S translation preinitiation complexes to persist throughout infection. These results indicate that in poliovirus-infected cells, the functions of TIA self-aggregation and aggregation of stalled translation initiation complexes into stress granules are severed, leading to novel foci that contain TIA1 but lack other stress granule-defining components. 相似文献
17.
Eukaryotic mRNA initiates translation by cap-dependent scanning, ribosome shunting and cap-independent internal ribosome entry. Internal ribosome entry was first discovered for cytoplasmic RNA viruses but has also been identified for DNA viruses and cellular mRNAs. An internal ribosome entry site (IRES) directs internal binding of ribosomes and nucleates the formation of a translation initiation complex. Current research is aimed at identifying interactions between IRES elements and RNA-binding proteins known as ITAFs (IRES trans-acting factors). Here we compare IRES elements from cytoplasmic RNA viruses with those of cellular mRNAs and DNA viruses with nuclear mRNA synthesis, and suggest that ITAF composition and IRES function directly reflect the site of synthesis of mRNA and the history of its pathway to polysomes. 相似文献
18.
The eukaryotic translation initiation factor 1 binds to the ribosome during translation initiation. It is instrumental for initiator-tRNA and mRNA binding, and has a function in selection of the authentic start codon. Here, we show that the archaeal homolog aIF1 has analogous functions. The aIF1 protein of the archaeon Sulfolobus solfataricus is bound to the small ribosomal subunit during translation initiation and accelerates binding of initiator-tRNA and mRNA to the ribosome. Accordingly, aIF1 stimulated translation of an mRNA in a S. solfataricus in vitro translation system. Moreover, this study suggested that the C terminus of the factor is of relevance for its function. 相似文献
19.
20.
eIF3: a versatile scaffold for translation initiation complexes 总被引:1,自引:0,他引:1
Hinnebusch AG 《Trends in biochemical sciences》2006,31(10):553-562
Translation initiation in eukaryotes depends on many eukaryotic initiation factors (eIFs) that stimulate both recruitment of the initiator tRNA, Met-tRNA(i)(Met), and mRNA to the 40S ribosomal subunit and subsequent scanning of the mRNA for the AUG start codon. The largest of these initiation factors, the eIF3 complex, organizes a web of interactions among several eIFs that assemble on the 40S subunit and participate in the different reactions involved in translation. Structural analysis suggests that eIF3 performs this scaffolding function by binding to the 40S subunit on its solvent-exposed surface rather than on its interface with the 60S subunit, where the decoding sites exist. This location of eIF3 seems ideally suited for its other proposed regulatory functions, including reinitiating translation on polycistronic mRNAs and acting as a receptor for protein kinases that control protein synthesis. 相似文献