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H O Voorma

Publications and source records attributed to H O Voorma.

At least 19 recordsLinked to original sources
PubMed1992

Interaction of initiation factors with the cap structure of chimaeric mRNA containing the 5'-untranslated regions of Semliki Forest virus RNA is related to translational efficiency.

Chimaeric chloramphenicol acetyltransferase (CAT) mRNA, containing the leader sequences of genomic 42S RNA and subgenomic 26S RNA of Semliki Forest virus (SFV) were synthesized by in-vitro transcription. These transcripts were translated with different efficiencies, as the authentic mRNA in SFV-infected cells. Therefore, they can be used as model mRNA species to study the mechanism underlying SFV-directed shut off of host protein synthesis. The interaction of translation initiation factors with the 5' cap structure was studied. Transcripts prepared in vitro using T7 RNA polymerase were capped and methylated posttranscriptionally with [32P]-GTP and S-adenosyl-L-methionine to yield cap-labelled mRNA species. Irradiation with ultraviolet light of 26S CAT and 42S CAT transcripts, together with crude rabbit reticulocyte initiation factors, resulted in the cap-specific cross-linking of eukaryotic initiation factors (eIF) eIF-4E and eIF-4B. The relative binding efficiency of these two factors to the cap structure of the various transcripts was, however, markedly different; the cap structure present in 26S CAT mRNA interacted efficiently with cap-binding proteins, whereas the cap structure of 42S CAT mRNA hardly bound to these proteins. Comparable results were obtained under competitive conditions. Data are presented that the secondary structure close to the 5' cap structure determines the efficiency of recognition of the mRNA by these initiation factors. Using a chemical cross-linking assay, it was demonstrated that eIF-4F, and also eIF-4E, differentially interacted with the cap structure of the various transcripts. The data are discussed with respect to the possible mechanisms involved in SFV-induced shut off of host cell protein synthesis.

Base Sequence↗
PubMed1992

Dependence of the adenovirus tripartite leader on the p220 subunit of eukaryotic initiation factor 4F during in vitro translation. Effect of p220 cleavage by foot-and-mouth-disease-virus L-protease on in vitro translation.

The adenovirus tripartite leader (TPT) 5' untranslated region (5'UTR) allows translation in poliovirus-infected cells, in which the p220 subunit of eukaryotic initiation factor 4F is degraded. This p220-independent translation was investigated by measuring in vitro translation in a reticulocyte lysate of a reporter gene, chloramphenicol acetyltransferase, coupled to the TPT 5'UTR. The p220 subunit was degraded by translation of a foot-and-mouth-disease L-protease construct. Surprisingly, the TPT 5'UTR was dependent on intact p220, as are other naturally capped mRNA species. Translation of encephalomyocarditis virus RNA was p220 independent, as expected from its ability to support internal, cap-independent initiation. In vitro protein-synthesis experiments with purified initiation factors confirmed the dependence of TPT mRNA translation on eukaryotic initiation factor 4F. The relationship between adenovirus TPT-5'UTR-directed translation and poliovirus-induced host cell shut-off is discussed.

Adenoviridae↗
PubMed1992

Eukaryotic initiation factors-4E and -4F stimulate 5' cap-dependent as well as internal initiation of protein synthesis.

Two mechanisms of initiation of protein synthesis are known. The 5' cap-dependent model requires the activity of cap-binding eukaryotic initiation factors (eIF)1-4E and -4F, inducing unwinding of mRNA secondary structures. The internal initiation model is 5' cap-independent and requires a ribosomal entry site formed by higher order structures of the mRNA. It has been proposed that this mechanism does not need eIF-4E and eIF-4F. We prepared bicistronic transcripts on which both mechanisms of initiation occur, allowing the determination of the initiation factor dependence of these two mechanisms simultaneously. The unwinding factors eIF-4A, eIF-4B, and eIF-4F were found to be necessary for 5' cap-dependent initiation as well as for internal initiation. Surprisingly, efficient translation of both cistrons on the bicistronic mRNA required eIF-4E. A model is presented in which assembly of eIF-4E into a functional eIF-4F complex is a prerequisite for both types of initiation.

Bacterial Proteins↗
PubMed1992

Interaction of protein synthesis initiation factors with the mRNA cap structure.

The mechanism of mRNA recognition by proteins interacting with the mRNA cap structure was investigated by photochemical cross-linking of proteins with 32P-labelled reoviral RNAs. Using ribosomal washes as a source of eukaryotic protein synthesis initiation factors, we identified the well-known cap binding proteins eIF-4B and -4E, but eIF-2 and eIF-3 as well. The interplay of purified eIF-4A, -4B, and -4F was studied in relation to ATP dependence and cap analogue sensitivity of cap binding. Next to their well-known roles in the initiation process, eIF-2 and eIF-3 also cross-linked to the 5' cap. eIF-2 stimulated eIF-4B and -4E cross-linking, an observation that has been previously described more extensively. The interaction of eIF-2 with the 5' end of mRNA was extremely sensitive to K(+)-ions and was resistant to a high concentration of Mg(2+)-ions; this influence of mono- and divalent ions was in contrast with the cross-linking of eIF-4B and -4E. Optimal interaction of these factors was obtained at moderate K+ concentration and low Mg(2+)-ion concentrations. eIF-2 cross-linking was sensitive to high protein to mRNA ratios indicating a weak affinity as compared to eIF-4E and -4B. The interaction of eIF-3 with the cap of mRNA is also weak as it was counteracted by all other cap binding proteins, leading to an inability to detect the cross-linking of this protein in crude eIF preparations. Time kinetics of formation of complexes suggested eIF-2 to be one of the first factors to interact with mRNA. Preformed RNA-protein complexes were dissociated after cap analogue addition, suggesting reversible interactions between RNA and proteins.

Adenosine Triphosphate↗
PubMed1992

RNA unwinding by eukaryotic initiation factor 4A and nucleotide modification.

Unwinding of double-stranded RNA by nuclear helicases can lead to modification of adenosine-residues, resulting in inosine. During initiation of protein synthesis the 5' untranslated region of an mRNA is unwound by eukaryotic initiation factors (eIF) -4A and -4B. In this work we investigated the possible nucleotide modification after unwinding by eIF-4A and eIF-4B of in vitro synthesized, labeled RNA. The products of unwinding were analyzed by gel-electrophoresis and, after nuclease digestion, by thin layer chromatography of the mononucleotides. Crude protein fractions unwound the duplex RNA and converted part of the AMP-residues into IMP-residues. However, unwinding by purified factors was not linked to this conversion, the deamination of AMP residues. Concluding, unwinding of RNA during initiation of protein synthesis does not lead to conversion of adenosine into inosine.

Base Sequence↗
PubMed1991

The 5' untranslated region of encephalomyocarditis virus contains a sequence for very efficient binding of eukaryotic initiation factor eIF-2/2B.

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.

Base Sequence↗
PubMed1991

Evidence that eukaryotic initiation factor (eIF) 2 is a cap-binding protein that stimulates cap recognition by eIF-4B and eIF-4F.

We studied the mRNA-binding properties of eukaryotic initiation factor (eIF) 2. This Met-tRNA-binding factor interacts with the cap structure of reoviral mRNA in an ATP-independent manner. Both the beta- and gamma-subunit of eIF-2 are involved in the UV-induced cross-linking of eIF-2 to the cap. The interaction of eIF-2 with a messenger is sensitive to the cap analogue 7-methyl-guanosine 5'-triphosphate as measured by cross-linking and by mRNA retention on nitrocellulose filters. The cap-binding property of eIF-2 does not conflict with the current mRNA-binding model of initiation factors eIF-4A, -4B, and -4F: cross-linking of eIF-4E and of eIF-4B is stimulated by eIF-2. The eIF-2-mediated increase of eIF-4E interaction results in a decrease of the cross-linking of the beta- and gamma-subunits of eIF-2. The presence of GTP in the cross-linking assay interferes with the interaction of eIF-2 with the cap structure but does not inhibit the eIF-2 stimulated eIF-4E and -4B cross-linking. These observations indicate a role for eIF-2 in the mRNA recognition.

Animals↗
PubMed1991

Separation of translationally active mRNAs by reversed-phase ion-pair high-performance liquid chromatography.

An ion-pair high-performance liquid chromatographic method on C4 columns was developed for the separation of mRNAs. The addition of methylmercuric hydroxide markedly influenced the separation according to length of these molecules. A method is given to recover minute amounts of translatable mRNA from the organic phase. The resolution of mRNAs improved with increasing pore size of the column support.

Animals↗
PubMed1979

A comparison of the initiation factors of eukaryotic protein synthesis from ribosomes and from the postribosomal supernatant.

A purification procedure is described for the initiation factors of protein synthesis from rabbit reticulocytes: (a) from the ribosomal wash and (b) from the postribosomal supernantant. A comparison is made between these preparations with respect to yield and specific activity. eIF-4A and eIF-4D occur mainly in the postribosomal supernatant; eIF-2, eIF-4C and eIF-5 are more evenly divided over both fractions, whereas eIF-1, eIF-3 and eIF-4B are found almost exclusively in the ribosomal wash. No significant difference in specific activity could be detected when factors from both sources were compared, with a possible exception of eIF-4A and eIF-4D.

Animals↗
PubMed1979

Purification and characterization of a protein factor that reverses the inhibition of protein synthesis by the heme-regulated translational inhibitor in rabbit reticulocyte lysates.

We have purified and partially characterized a supernatant factor which reverses the effect of the heme-regulated translational inhibitor on protein synthesis in rabbit reticulocyte lysates. The anti-inhibitor restores protein synthesis activity in heme deficient lysates (and in lysates to which the inhibitor has been added) to the level observed in the presence of heme. The factor has no effect on the phosphorylation of eIF-2 by the inhibitor nor on any reaction carried out with purified initiation factors. The anti-inhibitor probably consists of three subunits with molecular weights of 81000, 60000 and 41000. The factor is isolated from the postribosomal supernatant of rabbit reticulocytes both free and complexed to eIF-2. A possible mechanism of action is discussed.

Animals↗
PubMed1979

Postribosomal complexes containing eukaryotic initiation factor eIF-2.

Eukaryotic initiation factors are found in the post-ribosomal subunits. We have analyzed the factor activities from the supernatant by means of zonal centrifugation followed by Sepharose-heparin affinity chromatography. They exist both as free factors, sedimenting in a broad range from 4 to 7S, and complexed with other protein(s) with a sedimentation value of 16-20S. This complexed fraction contains besides eIF-2 another activity which exhibits a profound stimulation on amino acid incorporation in crude lysates and appears to counteract the heme-regulated inhibitor.

Animals↗
PubMed1979

The activity of eukaryotic initiation factor eIF-2 in ternary complex formation with GTP and Met-tRNA.

Ternary complex formation between [3H]Met-tRNAf, [14C]H3-eIF-2, and GTP was measured on nitrocellulose filters. It is shown that [3H]Met-tRNAf and [14C]H3-eIF-2 are present on the filter in equimolar amounts when ATP, creatine phosphate, and creatine kinase are included in the reaction mixture. Under these conditions the factor is 100% active. With small amounts of factor significant losses occur due to adsorption to the wall of the reaction vessels, resulting in seemingly low activities of eIF-2. These losses can be prevented by the presence of "stimulatory" proteins, which enhance the recovery of both [3H]Met-tRNAf and [14C]H3-eIF-2 on the filter but do not alter their ratio.

Animals↗
PubMed1978

Initiation factor IF-3 and the binary complex between initiation factor IF-2 and formylmethionyl-tRNA are mutually exclusive on the 30-S ribosomal subunit.

The formation of 30-S initiation complexes depends strongly on initiation factor IF-3; at molar ratios of IF-3 to 30-S ribosomes up to one a stimulation is observed, whereas at ratios higher than one, initiation complex formation declines strongly. The target of the observed inhibition of fMet-tRNA binding at high concentrations of IF-3 is the 30-S initiation complex itself. On the one hand addition of IF-3 to preformed 30-S initiation complexes leads to a release of bound fMet-tRNA which is linear with the amount of factor added, whereas no effect on isolated 70-S initiation complexes is seen. The release of fMet-tRNA from preformed 30-S initiation complexes is accompanied by a release of IF-2 in a one-to-one molar ratio which is in agreement with our previous findings showing that binding of fMet-tRNA takes place via a binary complex: IF-2 . fMet-tRNA (Eur. J. Biochem. 66, 181--192 and 77, 69--75). On the other hand increasing amounts of both IF-2 and fMet-tRNA relieve the IF-3-induced inhibition of 30-S initiation complex formation. From these findings it is concluded that IF-3 and the IF-2 . fMet-tRNA complex are mutually exclusive on the 30-S ribosome. This implies that under our experimental conditions MS2 RNA binding precedes fMet-tRNA binding if one accepts that the presence of IF-3 on the 30-S subunit is obligatory for messenger binding.

Escherichia coli↗