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W C Merrick

Publications and source records attributed to W C Merrick.

At least 55 records · Page 3Linked to original sources
PubMed1991

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

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↗
PubMed1990

Eukaryotic protein elongation factors.

In eukaryotes, peptide chain elongation is mediated by elongation factors EF-1 and EF-2. EF-1 is composed of a nucleotide-binding protein EF-1 alpha, and a nucleotide exchange protein complex, EF-1 beta gamma, while EF-2 catalyses the translocation of peptidyl-tRNA on the ribosome. Elongation factors are highly conserved among different species and may be involved in functions other than protein synthesis, such as organization of the mitotic apparatus, signal transduction, developmental regulation, ageing and transformation. Yeast contains a third factor, EF-3, whose structure and function is not yet well understood.

Animals↗
PubMed1990

Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F.

The mechanism of ribosome binding to eucaryotic mRNAs is not well understood, but it requires the participation of eucaryotic initiation factors eIF-4A, eIF-4B, and eIF-4F and the hydrolysis of ATP. Evidence has accumulated in support of a model in which these initiation factors function to unwind the 5'-proximal secondary structure in mRNA to facilitate ribosome binding. To obtain direct evidence for initiation factor-mediated RNA unwinding, we developed a simple assay to determine RNA helicase activity, and we show that eIF-4A or eIF-4F, in combination with eIF-4B, exhibits helicase activity. A striking and unprecedented feature of this activity is that it functions in a bidirectional manner. Thus, unwinding can occur either in the 5'-to-3' or 3'-to-5' direction. Unwinding in the 5'-to-3' direction by eIF-4F (the cap-binding protein complex), in conjunction with eIF-4B, was stimulated by the presence of the RNA 5' cap structure, whereas unwinding in the 3'-to-5' direction was completely cap independent. These results are discussed with respect to cap-dependent versus cap-independent mechanisms of ribosome binding to eucaryotic mRNAs.

Animals↗
PubMed1990

Overview: mechanism of translation initiation in eukaryotes.

Evidence to date has placed considerable emphasis on protein synthesis initiation as the dominant site of translational control. Two specific aspects are regulated, the binding of the initiator tRNA to the 40S subunits (as a ternary complex with eIF-2 and GTP) and the subsequent binding of mRNA to the complex of the 40S subunit with initiator tRNA. In addition to regulation, eIF-2 and Met-tRNAf are in large part responsible for selection of the initiating AUG codon. The utilization of most host mRNAs requires an m7G cap structure at the 5' end. However, many viral systems appear to use one of two alternate initiation schemes referred to as re-initiation and internal initiation. The function of specific initiation factors is presented and the consequences of altering the activity of these factors is discussed.

Gene Expression Regulation↗
PubMed1990

Translation initiation factors induce DNA synthesis and transform NIH 3T3 cells.

Several polypeptide factors that are essential for the initiation of protein synthesis bind to eukaryotic mRNAs and facilitate the formation of ribosome initiation complexes. Purified mRNA-binding translation initiation factors were microinjected into quiescent NIH 3T3 cells to study the possible growth-promoting role of these factors in living cells. We report that recombinant eIF-4E and rabbit reticulocyte eIF-4F induce a dose-dependent increase of DNA synthesis and morphologically transform NIH 3T3 cells. These results suggest that polypeptides involved in activating the rate-limiting step of protein synthesis (initiation complex formation) can be mitogenic and oncogenic when overexpressed in a cell by direct injection. Thus, eIF-4E and eIF-4F represent a class of proto-oncogenic proteins that is cytoplasmic, is involved in protein synthesis initiation, and is distinct from the proto-oncogenes that have been identified previously.

Animals↗
PubMed1989

Location of seven post-translational modifications in rabbit elongation factor 1 alpha including dimethyllysine, trimethyllysine, and glycerylphosphorylethanolamine.

Amino acid sequencing of a large number of chemical and enzymatic cleavage products of elongation factor 1 alpha purified from rabbit reticulocyte has identified seven post-translationally modified residues. Five of the modifications are methylations of lysine residues yielding dimethyllysine at residues 55 and 165 and trimethyllysine at residues 36, 79, and 318. The two remaining post-translational modifications involve the addition of ethanolamine to glutamic acid residues 301 and 374, as reported previously (Rosenberry, T. L., Krall, J. A., Dever, T. E., Haas, R., Louvard, D., and Merrick, W. C. (1989) J. Biol. Chem. 264, 7096-7099). Fast atom bombardment mass spectrometry and fast atom bombardment tandem mass spectrometry have been used to analyze peptides containing these modified residues. The analyses have determined that glycerylphosphorylethanolamine has been attached to the glutamic acid residues. An analysis of the amino acid sequence surrounding each of the three types of modification has indicated no similarities. Therefore, it seems likely that the modifying enzymes do not recognize a specific amino acid sequence but rather the three-dimensional presentation of either amino or carboxyl residues in the elongation factor 1 alpha structure.

Amino Acid Sequence↗
PubMed1989

Dissociation of double-stranded polynucleotide helical structures by eukaryotic initiation factors, as revealed by a novel assay.

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.

Chromatography, Gel↗
PubMed1989

Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1 alpha reveals a new post-translational protein modification.

Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleukemia cell line K562. A single predominant labeled protein of about 50 kDa was observed following electrophoresis of cell extracts on polyacrylamide gels in the presence of sodium dodecyl sulfate. Subcellular fractionation showed this protein to distribute similarly to a 46-kDa [3H]ethanolamine-labeled protein reported previously (Tisdale, E. J., and Tartakoff, A. M. (1988) J. Biol. Chem. 263, 8244-8252). In particular, the protein was enriched in cytosolic and microsomal fractions relative to plasma membrane and thus did not appear to correspond to the class of proteins with glycoinositol phospholipid anchors, the only post-translational protein modification involving ethanolamine that had been described previously. Two-dimensional polyacrylamide gel analysis involving isoelectric focusing followed by electrophoresis in sodium dodecyl sulfate indicated that the protein was very basic, and nitrocellulose blots of one- and two-dimensional gels subjected to 3H autoradiography and immunostaining with antisera to purified rabbit elongation factor (EF) 1 alpha revealed that the protein was EF-1 alpha. Copurification of rabbit EF-1 alpha and the [3H]ethanolamine-labeled protein from K562 cells further supported this identification. Analysis of tryptic fragments produced from the copurified proteins by reverse-phase high pressure liquid chromatography showed two radiolabeled peptides. Amino acid analysis demonstrated 1 residue of ethanolamine in each peptide, and peptide sequencing revealed that the ethanolamine-containing component(s) was attached to Glu301 and Glu374 in the EF-1 alpha protein sequence deduced from a human EF-1 alpha cDNA. These data confirm a new class of post-translational protein modifications involving ethanolamine.

Animals↗
PubMed1989

Comparative analysis of phosphorylation of translational initiation and elongation factors by seven protein kinases.

Four initiation factors (eIF-2, -3, -4B, and -4F), previously shown to be phosphorylated in vivo, are each phosphorylated to a significant extent in vitro (greater than 0.3 mol of phosphate/mol of factor) by at least three different protein kinases. An S6 kinase from liver, an active form of protease-activated kinase II which modifies the same sites on S6 as those phosphorylated in vivo in response to mitogens, phosphorylates the beta subunit of eIF-2, eIF-3 (p120-p130), eIF-4B, and eIF-4F (p220). The Ca2+, phospholipid-dependent protein kinase phosphorylates eIF-2 beta, eIF-3 (p170, p120-p130), eIF-4B, and eIF-4F (p220, p25). The cAMP-dependent protein kinase significantly modifies eIF-4B and, to a lesser extent, eIF-3 (p130). Casein kinase I incorporates phosphate only into eIF-4B, but to a limited extent. Casein kinase II phosphorylates eIF-2 beta, eIF-3 (p170, p120), and eIF-4B, while protease-activated kinase I modifies eIF-3 (p170, p120-p130), eIF-4B, and eIF-4F (p220). The mitogen-stimulated S6 kinase from 3T3-L1 cells, activated in response to insulin, does not phosphorylate any of the initiation factors. There is no significant incorporation of phosphate into eIF-2 alpha or -gamma, eIF-4A, eIF-4C, eIF-4D, EF-1, or EF-2 by any of the protein kinases examined. Phosphopeptide mapping of tryptic digests of the phosphorylated subunits shows that the individual protein kinases modify different sites. The sites phosphorylated in vitro reflect those modified in vivo as shown with eIF-4F in concomitant studies with reticulocytes treated with tumor-promoting phorbol ester (Morley, S.J., and Traugh, J. A. J. Biol. Chem., in press). Thus, we have identified multipotential protein kinases which modify four initiation factors phosphorylated in vivo and have shown that phosphorylation of these translational components can be coordinately regulated.

Kinetics↗