Nucleotide sequence of rabbit elongation factor 1 alpha cDNA.
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Amino Acid Sequence↗
Biomedical subjects
W C Merrick
Publications and source records attributed to W C Merrick.
Two isoforms of eIF-5A in chick embryo. Isolation, activity, and comparison of sequences of the hypusine-containing proteins.
Eukaryotic translation initiation factor 5A (eIF-5A) (older terminology, eIF-4D) is unique in that it contains the unusual amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Hypusine is formed by a post-translational event in which a specific lysine residue is modified by a structural contribution from spermidine. Metabolic labeling of chick embryo fibroblasts with [3H]spermidine or [3H]lysine gives rise to two distinct proteins, designated I (approximately 20 kDa and pI 5.6) and II (approximately 18 kDa and pI 5.35), that contain [3H]hypusine. Upon incubation with [3H]lysine the labeling of the two proteins followed a similar time course and showed approximately the same ratio over the 6-h incubation period. [3H]Hypusine-containing proteins from cells which had been cultured with [3H]spermidine were employed as tracers for isolation of hypusine-containing proteins from whole chick embryos. Four such proteins were obtained. Two of these proteins, I and II, correspond to the two native proteins synthesized in chick embryo fibroblasts; the other two forms, Ia and IIa, displayed properties suggesting that they were derived from the native proteins, I and II, respectively, during purification. The amino acid compositions and the tryptic peptide maps of the 20-kDa protein (I) and the 18 kDa protein (II) suggest that they are closely related but distinct proteins. In fact, amino acid sequence analysis of the two major proteins revealed differences in the polypeptide backbone of the two proteins. In spite of structural differences, the two native forms (I and II), as well as the two altered forms (Ia and IIa), were effective in stimulating methionyl-puromycin synthesis, providing evidence that they are indeed functional isoforms of eIF-5A.
Amino Acid Sequence↗
Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition.
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.
Adenosine↗
A model for the aminoacyl-tRNA binding site of eukaryotic elongation factor 1 alpha.
Eukaryotic elongation factor 1 alpha (EF-1 alpha) binds all the aminoacyl-tRNAs except the initiator tRNA in a GTP-dependent manner. While the GTP binding site is delineated by the three GTP binding consensus elements, less is known about the aminoacyl-tRNA binding sites. In order to better understand this site, we have initiated cross-linking and protease mapping studies of the EF-1 alpha-GTP-aminoacyl-tRNA complex. Two different chemical cross-linking reagents, trans-diaminedichloroplatinum(II) and diepoxybutane, were used to cross-link four different aminoacyl-tRNA species to EF-1 alpha. A series of peptides were obtained, located predominantly in domains II and III. The ability of aminoacyl-tRNA to protect protease digestion sites was also monitored, and domain II was found to be protected from digestion by aminoacyl-tRNA. Last, an aminoacyl-tRNA analog with a reactive group on the aminoacyl side chain, N epsilon-bromoacetyl-Lys-tRNA, was cross-linked to EF-1 alpha. This reagent cross-liked to histidine 296 in a GTP-dependent manner and thus localizes the aminoacyl group adjacent to domain II. A model is developed for aminoacyl-tRNA binding to EF-1 alpha based on its similarity to the prokaryotic factor EF-Tu, for which an x-ray crystal structure is available.
Amino Acid Sequence↗
Mechanism and regulation of eukaryotic protein synthesis.
This review presents a description of the numerous eukaryotic protein synthesis factors and their apparent sequential utilization in the processes of initiation, elongation, and termination. Additionally, the rare use of reinitiation and internal initiation is discussed, although little is known biochemically about these processes. Subsequently, control of translation is addressed in two different settings. The first is the global control of translation, which is effected by protein phosphorylation. The second is a series of specific mRNAs for which there is a direct and unique regulation of the synthesis of the gene product under study. Other examples of translational control are cited but not discussed, because the general mechanism for the regulation is unknown. Finally, as is often seen in an active area of investigation, there are several observations that cannot be readily accommodated by the general model presented in the first part of the review. Alternate explanations and various lines of experimentation are proposed to resolve these apparent contradictions.
Animals↗
Eukaryotic initiation factor (eIF)-4F. Implications for a role in internal initiation of translation.
In order to study the eukaryotic translation initiation mechanisms of "internal initiation," "re-initiation," and/or "coupled internal initiation," a series of model mRNAs have been constructed which contain two non-overlapping open reading frames (ORFs) that encode different lengths of rabbit alpha globin. These mRNAs, along with the bicistronic constructs TK/CAT and TK/P2CAT developed by Pelletier and Sonenberg (Pelletier, J., and Sonenberg, N. (1988) Nature 334, 320-325, 1988), were used to program an in vitro rabbit reticulocyte lysate translation system. Cap-dependent and cap-independent translation were distinguished by monitoring translation in the presence or absence of exogenously added cap analog (m7GTP). Messenger RNAs which translate both ORF1 and ORF2 by a cap-dependent mechanism, as well as mRNAs that translate ORF2 by a cap-independent mechanism while still translating ORF1 in a cap-dependent fashion have been obtained. These same alpha globin mRNAs differ by no more than 45 nucleotides in intercistronic length. Initiation factor addition studies were performed in this same in vitro translation system. Both eukaryotic initiation factor (eIF)-4F and, to a lesser extent, eIF-4B can stimulate translation of an internally located ORF independent of upstream ORF translation and in a manner not dependent on mRNA cap recognition. This indicates that the cap-recognition initiation factor, eIF-4F, and eIF-4B facilitate cap-independent and internal initiation of an open reading frame.
Animals↗
Characterization of a limited trypsin digestion form of eukaryotic elongation factor 1 alpha.
The involvement of the first 69 amino acids of eukaryotic elongation factor 1 alpha (EF-1 alpha) from rabbit reticulocyte in GTP and aminoacyl-tRNA binding has been analyzed by a variety of techniques. EF-1 alpha was subjected to limited trypsin digestion, which cleaved predominantly at residues 36 and 69. A digested form of Escherichia coli EF-Tu, similar to the one used for this study, has been characterized by x-ray crystallography and is used as a structural model for EF-1 alpha. This form of EF-1 alpha bound E. coli Phe-tRNAPhe similar to the wild type protein, but lacked activity in phenylalanine polymerization with poly(U)-programmed ribosomes. These results were obtained regardless of whether or not loosely associated N-terminal peptides were removed by gel filtration chromatography. The digested EF-1 alpha also shows reduced GTPase activity, but the activity is stimulated by both ribosomes and aminoacyl-tRNA. Binding of EF-1 alpha to the 80 S ribosome, as determined by association of reductively methylated protein through Sepharose 6B chromatography, is reduced approximately 7-fold for the limited digested form of the protein. Limited digested EF-1 alpha can, however, be photo-cross-linked with GTP and 3'-p-azido-GTP similar to intact EF-1 alpha. Chemical cross-linking with oxidized GTP, fluorosulfonylbenzoyl-GTP, or with trans-diaminedichloroplatinum(II) and GPT, shows a similar modification of both intact and limited digested EF-1 alpha. In order to further localize the modification site with the GTP reagents and assure that modification was not occurring in the first 69 amino acids, intact EF-1 alpha was modified with these same reagents. Limited trypsin digestion of modified protein indicates that none of these reagents cross-links GTP to the first 69 amino acids of EF-1 alpha, which includes the first GTP binding consensus element, GXXXXGK.
Affinity Labels↗
Effect of eukaryotic initiation factor 4F on AUG selection in a bicistronic mRNA.
Artificial bicistronic mRNAs based on rabbit beta-globin and bacterial chloramphenicol acetyltransferase protein-coding sequences were tested for translation activity in a mouse astrocytoma cell-free extract. This cell extract exhibited an apparent preference for 5'-distal or internal initiation over 5'-proximal ("first AUG") initiation. 5'-Distal initiation appeared to be 5'-cap independent, suggesting that nonstandard initiation was responsible. This conclusion was based on a lack of inhibition of internal initiation by added cap analog and insensitivity of internal initiation to the presence or absence of a 5'-cap structure. Exogenous reticulocyte initiation factors were tested for effect on 5'-proximal initiation. The only factor with a significant effect was found to be eukaryotic initiation factor 4F, or the cap-binding protein. Addition of this factor promoted 5'-end initiation as evident by a general increase in 5'-proximal open reading frame (ORF) product relative to 5'-distal ORF product. The relative expression of 5'-proximal to 5'-distal ORFs in bicistronic or multicistronic mRNAs may very well be dependent on activity levels of eukaryotic initiation factor 4F and possibly other mRNA-dependent initiation factors.
Animals↗
Mechanism of selective translation of vaccinia virus mRNAs: differential role of poly(A) and initiation factors in the translation of viral and cellular mRNAs.
We have recently demonstrated that the poly(A) moieties of short RNAs obtained from both in vitro transcription and from vaccinia virus (VV)-infected cells exhibit dissimilar effects on the in vitro translation of cellular and VV mRNAs (R. Bablanian, G. Coppola, P. Masters, and A. K. Banerjee, Virology 148:375-380, 1986; M. J. Su and R. Bablanian, Virology 179:679-693, 1990). In the present study, we have investigated the roles of poly(A), m7GTP, and initiation factors in the mechanism of selective translation of VV mRNAs. The effects of unfractionated poly(A) [termed poly(A)un, with various chain lengths up to 3,000 nucleotides] and a 150- to 300-nucleotide fraction of synthetic poly(A) [termed poly(A)150-300] on the translation of HeLa cell mRNAs and early and late VV mRNAs were studied. Both the poly(A)un and the poly(A)150-300 completely inhibited the translation of HeLa cell mRNAs obtained from total cytoplasmic RNA in the nuclease-treated reticulocyte lysates. Viral mRNAs from total cytoplasmic RNA also were slightly inhibited (15 to 38%) by the poly(A)un, whereas the poly(A)150-300 had no significant effect on their translation. The translation of oligo(dT)-cellulose-selected HeLa mRNAs was as sensitive to inhibition by poly(A)150-300 as the mRNAs found in total cytoplasmic RNA. However, the translations of oligo(dT)-cellulose-selected viral mRNAs become more sensitive to the inhibitory effect of poly(A)150-300 than the translations of viral mRNAs found in the total cytoplasmic RNA. Both HeLa and VV mRNAs became more resistant to the poly(A)-mediated inhibition when these mRNAs were deadenylated, but the relative resistance to inhibition by poly(A)150-300 of deadenylated VV mRNAs was much greater than that of HeLa cell mRNAs. The translation of VV mRNAs was significantly less inhibited than the translation of HeLa mRNAs when the cap analog, m7GTP, was added to the cell-free system. The inhibition of HeLa cell mRNA translation by both poly(A)un and poly(A)150-300 was completely restored when poly(A)-binding protein (PAB) was added to the cell-free translational system. The addition of eukaryotic initiation factor 4A (eIF-4A) did not restore translation when poly(A)un was used to inhibit translation; however, inhibition by poly(A)150-300 was significantly reversed by this initiation factor. The reversal of poly (A)-mediated inhibition of HeLa cell mRNA translation was additive when PAB was used together with eIF-4A. Early VV mRNA translation was only slightly inhibited by poly(A)un (15%), and this inhibition was completely reversed by either PAB or eIF-4A.(ABSTRACT TRUNCATED AT 400 WORDS)
Carrier Proteins↗
RNA unwinding in translation: assembly of helicase complex intermediates comprising eukaryotic initiation factors eIF-4F and eIF-4B.
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.
Base Sequence↗
Everything you wanted to know about protein synthesis ... almost. Translational control: a Keystone symposium, Tamarron, CO, USA, February 22-March 1, 1991.
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Animals↗
Characterization of the 46,000-dalton subunit of eIF-4F.
Three protein synthesis initiation factors, eukaryotic initiation factor (eIF)-4A, -4B, and -4F are required for the ATP-dependent binding of mRNA to the ribosome. To extend the characterization of the eIF-4A-like subunit of eIF-4F, a cDNA clone encoding eIF-4A has been isolated from a rabbit liver cDNA library and sequenced. The clone is almost full length for the coding region and complete for the 3' noncoding region. The sequence of the rabbit cDNA has been compared to the sequence of the two similar, but not identical, genes and cDNAs encoding mouse eIF-4A (termed eIF-4AI and eIF-4AII). The rabbit cDNA sequence is very similar to the mouse eIF-4AI genomic and liver cDNA sequence with 100% identity at the amino acid level and 90% identity at the nucleotide level within the protein coding region; however, there is very little similarity in the 3' noncoding region. Amino acid sequencing of purified rabbit reticulocyte eIF-4A protein indicates that it is eIF-4AI (encoded by the eIF-4AI gene and cDNA) and none of the amino acid residues sequenced are in disagreement with those predicted from the mouse liver or rabbit liver cDNA sequences. Subsequently, we have analyzed the p46 subunit of eIF-4F, a three subunit protein whose molecular weights have been estimated by sodium dodecyl sulfate gel electrophoresis to be 220,000, 46,000 and 24,000. The p46 subunit has physical properties similar to eIF-4A. This subunit was isolated from rabbit reticulocyte eIF-4F and sequenced chemically. Our results indicate that this peptide is a mixture of eIF-4AI and eIF-4AII in an approximate ratio of 4 to 1, respectively. No eIF-4AII was observed in our rabbit reticulocyte eIF-4A preparation. Therefore we have concluded that either the eIF-4AI and the eIF-4AII proteins were resolved from each other in the purification of rabbit reticulocyte eIF-4A or that eIF-4AII preferentially associates with the p220 and p24 subunits of eIF-4F. Evidence favoring the latter possibility is discussed.
Amino Acid Sequence↗
Translation initiation factors that function as RNA helicases from mammals, plants and yeast.
Ribosome binding to eukaryotic mRNAs requires the concerted action of three eukaryotic initiation factors: eIF-4A, eIF-4B and eIF-4F as well as the hydrolysis of ATP. These initiation factors are implicated in the unwinding of mRNA 5' secondary structure and have been isolated from mammals, yeast and wheat germ. We used an RNA unwinding assay to compare the activities of these factors from the different species. We also measured the inter-species interchangeability of these factors in the unwinding reaction. In mammals, it has been previously shown that a combination of rabbit reticulocyte eIF-4F and -4B or eIF-4A and -4B were active in the RNA unwinding assay. In wheat germ, the combination of eIF-4A and eIF-4F resulted in RNA unwinding in a reaction that was stimulated by eIF-4B. Mammalian eIF-4A was able to substitute in this system. We also show that yeast eIF-4A is able to effectively substitute for mammalian eIF-4A in duplex RNA unwinding in combination with mammalian eIF-4B, while wheat-germ eIF-4A was only partially able to substitute. Taken together, these results suggest that initiation factor requirements for RNA unwinding are largely similar in mammals, yeast and plants.
Animals↗
A fluorescence study of the interaction of protein synthesis initiation factors 4A, 4E, and 4F with mRNA and oligonucleotide analogs.
The initial interaction of mRNA with the protein synthesis machinery presumably involves recognition of the 5'-cap (m7GpppN), although it is not clear at the present time whether this recognition is by eIF-4E or eIF-4F. This process has been studied by direct fluorescence titration experiments. The equilibrium constants for the formation of the binary protein: m7GpppG, protein:mRNA, and protein:protein complexes as well as the ternary mRNA:eIF-4E:eIF-4A complexes were measured. These studies show, for the first time, direct evidence for an eIF-4A:eIF-4E interaction. In contrast to earlier studies, we show that the affinity of eIF-4E and eIF-4F for globin mRNA is similar. Furthermore, the relative affinities of mRNA analogs (capped oligonucleotides) for these initiation factors indicate that the cap is the predominant feature recognized for binding, but other features also contribute to the eIF-4E:mRNA interaction.
Animals↗
Characterization of protein synthesis factors from rabbit reticulocytes.
As part of our efforts to characterize eukaryotic translation factors, we have sequenced a number of them chemically and inferred sequences from cDNA clones. To our surprise, there appears to be extensive identity of amino acid sequence in most factors characterized to date in that within mammalian species, usually greater than 99% identity is observed. Extreme examples are rabbit EF-1 alpha which is 100% identical to human EF-1 alpha and rabbit eIF-4AI and eIF-4AII which are 100% identical to mouse eIF-4AI and eIF-4AII for those amino acids sequenced (398/406 and 156/407, respectively). An extended analysis has been made of EF-1 alpha which in rabbit has three different post-translational modifications, dimethyllysine, trimethyllysine and glycerylphosphorylethanolamine. A comparison of the primary structure of EF-1 alpha to E. coli EF-Tu indicates an overall sequence identity of 33%. However, within the amino terminal 180 amino acids (the GTP-binding domain), there are found regions of much greater identity (50/85 = 59%).
Amino Acid Sequence↗
Affinity labeling of eukaryotic initiation factor 2 and elongation factor 1 alpha beta gamma with GTP analogs.
As part of an attempt to understand the specific function and role of each subunit in multisubunit protein synthesis factors, we have attempted to identify the nucleotide binding peptides of eukaryotic initiation factor 2 (eIF-2). To ensure that the interactions were of a specific nature, two general controls were used: first, other protein factors with characterized GTP binding activity were tested; second, all affinity labeling was checked for nucleotide specificity by protection with the authentic nucleotide at a 10-fold molar excess over the affinity reagent. Results with a number of GTP modifying reagents ([alpha-32P]GTP, [alpha-32P]GDP, oxidized [alpha-32P]GTP, 3'-p-azidobenzoyl-[alpha-32P]GTP, 3'-p-azidobenzoyl-[alpha-32P]GDP, and 5'-p-[8-3H]fluorosulfonylbenzoyl guanosine) indicate that appropriate conditions for both nucleotide and subunit specific labeling have been achieved. Under these conditions all reagents modified the beta subunit of eIF-2. Complementary studies with subunit-deficient forms of eIF-2 also suggest that the beta subunit of eIF-2 is involved with GTP binding. Coupled with other data suggesting that the gamma subunit of eIF-2 might be involved in GTP binding and amino acid sequence data of eIF-2 gamma from which a part of a GTP binding consensus sequence can be localized, support is provided for the concept of alternate GTP binding domains or a GTP binding domain shared between different subunits of eIF-2.
Affinity Labels↗
Association of initiation factor eIF-4E in a cap binding protein complex (eIF-4F) is critical for and enhances phosphorylation by protein kinase C.
Phosphorylation by protein kinase C of the mRNA cap binding protein purified as part of a cap binding protein complex (eIF-4F) or as a single protein (eIF-4E), has been examined. Significant phosphorylation (up to 1 mol of phosphate/mol of p25 subunit) occurs only when the protein is part of the eIF-4F complex. With purified eIF-4E, using the same conditions, up to 0.1 mol of phosphate can be incorporated. Tryptic phosphopeptide maps show that the site phosphorylated in the Mr 25,000 subunit of eIF-4F (eIF-4F p25) is the same as that modified in purified eIF-4E. Kinetic measurements obtained from initial rates indicate that the Km values for eIF-4F and eIF-4E are similar, although the Vmax is 5-6 times higher for the complex. Dephosphorylation of eIF-4F p25, previously phosphorylated with protein kinase C, occurs in reticulocyte lysate with a half-life of 15-20 min, whereas little dephosphorylation is observed after 15 min with the purified phosphorylated eIF-4E. Phosphorylation of eIF-4F on the p220 and p25 subunits does not affect the stability of the complex as indicated by gel filtration on Sephacryl S-300. However, addition of non-phosphorylated eIF-4E to the phosphorylated complex results in the dissociation of the complex. These results suggest that interaction of p25 with other subunits in the complex greatly affects phosphorylation/dephosphorylation of p25. Since the rate of phosphorylation/dephosphorylation is significantly greater in the complex, regulation of the cap binding protein by phosphorylation appears to occur primarily on eIF-4F.
Animals↗
Fluorescence study of the binding of m7GpppG and rabbit globin mRNA to protein synthesis initiation factors 4A, 4E, and 4F.
The interactions of protein synthesis initiation factors eIF-4E from human erythrocytes and eIF-4A and eIF-4F from rabbit reticulocytes with the cap analogue m7GpppG and rabbit globin mRNA were investigated. The equilibrium binding constants for the binary complex formation of eIF-4E-eIF-4A, m7GpppG-eIF-4E, m7GpppG-eIF-4F, globin mRNA-eIF-4E, globin mRNA-eIF-4F, and globin mRNA-eIF-4A were measured by direct fluorescence titration experiments. The binding of eIF-4E to globin mRNA was found to be 5.5-fold tighter than its binding to m7GpppG; the binding of eIF-4F for globin mRNA and m7GpppG was similar to that of eIF-4E. Association equilibrium constants were determined for the ternary system mRNA-eIF-4E-eIF-4A; four thermodynamically independent equilibria characterize the system. These equilibrium binding constants were used to calculate coupling free energies, which provided an estimate of the cooperativity of the interaction of eIF-4E, eIF-4A, and mRNA. These coupling energies were all found to be small and positive, indicative of anticooperative binding.
Binding Sites↗