Naba K. Gupta

Protein synthesis in rabbit reticulocytes: Characteristics of a Met-tRNAfMet binding factor

Abstract Three factors (IF 1 , IF 2 , IF 3 ) resolved by DEAE-cellulose column chromatography of the crude reticulocyte ribosomal salt wash (I fraction), stimulated poly r(U-G) directed methionine transfer from Met-tRNA f Met into the terminal positions of the synthesized polypeptides. All three factors were necessary for maximum methionine transfer. A protein factor that is also present in the I fraction and elutes similarly to IF 1 on DEAE-cellulose chromatography, binds Met-tRNA f Met in the presence of GTP. The complex formed is quantitatively retained on Millipore filter. This complex formation is specific for Met-tRNA f Met . Other amino acyl tRNAs tested such as Met-tRNA m Met , Phe-tRNA Phe and Val-tRNA Val do not form a similar complex. With Met-tRNA f Met , complex formation does not require Mg ++ ion or AUG codon and is inhibited by the addition of ribosomes.

Roles of methionine transfer RNA's in protein synthesis in rabbit reticulocytes☆

Abstract DEAE-Sephadex column chromatography of crude rabbit liver transfer RNA gives rise to three peaks of methionine-acceptor activity. Only one of these tRNAMet species (tRNAfMet) † could be charged by Escherichia coli synthetase and, as previously reported, could be formylated by E. coli formylase. The other two tRNAMet species (tRNAm1Met and tRNAm2Met) could not be charged by E. coli synthetase. The coding properties of the tRNAMet species were studied in a cell-free protein synthesizing system obtained from rabbit reticulocytes with poly r(A-U-G) messenger and endogenous messenger in reticulocyte ribosomes. These studies indicated that with rabbit reticulocytes, tRNAfMet recognizes specifically the terminal methionine codon (AUG) and transfers methionine to the N-terminal positions of the polypeptide chains while tRNAmMet recognizes specifically the internal methionine codon (AUG) and inserts methionine into the internal positions of the polypeptide chains.

Protein synthesis in rabbit reticulocytes: Control of protein synthesis initiation by a Met-tRNAfMet deacylase and peptide chain initiation factors

Abstract The 0.5M KCl wash of rabbit reticulocyte ribosomes (I fraction) catalyzes the deacylation of Met-tRNA f Met . Upon DEAE-cellulose column chromatography, the deacylase activity elutes with the 0.1M KCl wash of the column (f1) and is well-resolved from the peptide chain initiation factors (1–3). The deacylase activity is specific for Met-tRNA f Met (retic., E. coli ). Other aminoacyl tRNAs tested including fMet-tRNA f Met (retic., E. coli ), Phe-tRNA ( E. coli ), Val-tRNA (retic.), and Arg-tRNA (retic.) are completely resistant to the action of the deacylase. In the presence of the peptide chain initiation factor (IF1) and GTP, retic. Met-tRNA f Met forms the initiation complex Met-tRNA f Met :IF1:GTP (2), and in this ternary complex Met-tRNA f Met is not degraded by the deacylase. E. coli Met-tRNA f Met binds to IF1 independent of GTP, and in this complex, this Met-tRNA f Met is degraded by the deacylase. Prior incubation of f1 with Met-tRNA f Met (retic.) strongly inhibited protein synthesis initiation, presumably due to deacylation of the initiator tRNA. This inhibition by f1 was completely prevented when Met-tRNA f Met (retic.) was pre-incubated with peptide chain initiation factors.

Initiation of protein synthesis in rabbit reticulocytes

Abstract DEAF-Sephadex column chromatography of crude rabbit liver transfer RNA (tRNA) gives rise to three peaks of methionine acceptor activity (met-tRNAMI, met-tRNAMIL , and met-tRNAF).Only one of these species (met-tRNAF) could be charged by E. coli synt hetase and formylated by E coli formylase (1,2). In a cell-free protein synthesizing system obtained from rabbit reticulocytes, [14C] met-tRNAF transfers radioactivity to the N-terminal position of the polypeptide chain in response to native messenger RNA. In a similar experiment, [14C] met-tRNAMII transfers radioactivity mainly into the internal positions of the synthesized polypeptide chains.

Protein synthesis in rabbit reticulocytes. XV: Isolation of a ribosomal protein factor (CO-EIF-1) which stimulates Met-tRNAfMet binding to EIF-1☆

Abstract A protein factor, CO-EIF-1, has been partially purified from the high salt wash of reticulocyte ribosomes. CO-EIF-1, by itself, does not bind Met-tRNA f Met but significantly stimulates (2–3 fold) Met-tRNA f Met binding to EIF-1 and also increases the initial rate of this binding reaction. In the presence of CO-EIF-1, approximately 90 percent of the input EIF-1 (assuming a molecular weight of 140,000) was bound to Met-tRNA f Met .

A Eukaryotic Translation Initiation Factor 2-Associated 67 kDa Glycoprotein Partially Reverses Protein Synthesis Inhibition by Activated Double-Stranded RNA-Dependent Protein Kinase in Intact Cells

A eukaryotic translation initiation factor 2 (eIF-2)-associated 67 kDa glycoprotein (p67) protects the eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, and this promotes protein synthesis in the presence of active eIF-2 alpha kinases in vitro [Ray, M. K., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543]. We have now examined the effect of overexpression of this cellular eIF-2 kinase inhibitor in an in vivo system using transiently transfected COS-l cells. In this system, coexpression of genes that inhibit PKR activity restores translation of plasmid-derived mRNA. We now report the following. (1) Transient transfection of COS-1 cells with a p67 expression vector increased p67 synthesis by 20-fold over endogenous levels in the isolated subpopulation of transfected cells. (2) Cotransfection of p67 cDNA increased translation of plasmid-derived mRNAs. (3) Overexpression of p67 reduced phosphorylation of coexpressed eIF-2 alpha. (4) p67 synthesis was inhibited by cotransfection with an eIF-2 alpha mutant S51D, a mutant that mimics phosphorylated eIF-2 alpha, indicating that p67 cannot bypass translational inhibition mediated by phosphorylation of the eIF-2 alpha-subunit. These results show that the cellular protein p67 can reverse PKR-mediated translational inhibition in intact cells.

Protein synthesis in rabbit reticulocytes: Characteristics of peptide chain initiation factors

Summary In a cell-free amino acid incorporating system using preincubated reticulocyte ribosomes, the transfer of methionine from [ 35 S] Met-tRNA f Met in response to poly r((U-G) messenger (in the presence of unlabeled valine, cysteine and crude tRNA) was dependent on addition of the 0.5M KCl wash of crude reticulocyte ribosomes. The optimum Mg ++ concentration for this transfer reaction was 3mM. The ribosomal salt wash preparation was fractionated by DEAE-cellulose column chromatography. At least three factors (I 1 , I 2 and I 3 ) that eluted from the column stimulated poly r(U-G) directed methionine transfer from Met-tRNA f Met . All three factors were necessary to obtain maximum transfer of methionine. Preincubated reticulocyte ribosomes actively catalyzed the incorporation of [ 14 C] phenylalanine in response to poly r(U) and the addition of the ribosomal salt wash or the above three factors (I 1 , I 2 and I 3 ), had no significant effect on [ 14 C] phenylalanine incorporation.

New enzyme cycling method for determination of oxidized and reduced nicotinamide-adenine dinucleotide

A highly sensitive enzyme cycling method for the measurement of NAD+(H) concentration in tissue extracts has been described. The assay measured propanediol formed during the coupled oxidation-reduction reactions between ethanol and lactaldehyde as catalyzed by liver alcohol dehydrogenase in the presence of catalytic concentrations of NAD+(H). The assay can be used to determine NAD+(H) concentrations in the range of 0.4 to 4 pmoles of coenzyme in a 0.1 ml reaction volume.

Protein synthesis in rabbit reticulocytes XIX: EIF-2 promotes dissociation of Met-tRNAf-EIF-1-GTP complex and Met-tRNAf binding to 40S ribosomes.

Abstract The peptide chain initiation factor, EIF-2 has been partially purified from the 0.5 M KCl ribosomal wash. The molecular weight of EIF-2 is approximately 450,000. The purified EIF-2 preparation promotes the dissociation of the ternary complex, Met-tRNA f ·EIF-1·GTP in the presence of Mg ++ and is also required along with EIF-1 for AUG-directed Met-tRNA f binding to 40S ribosomes.

Protein synthesis in rabbit reticulocytes XIII: Lack of mRNA (poly r(A)) binding activity in highly purified EIF-1

Abstract Met-tRNA f Met binding factor (EIF-1) has been purified more than 100 fold over crude high salt (0.5 M KCl) ribosomal wash. The purified factor binds 2 nmoles Met-tRNA f Met per mg protein and shows very little poly r(A) binding activity. Crude ribosomal high salt wash possesses significant amounts of poly r(A) binding activity and also binds to other RNAs. The bulk of this unspecific RNA binding protein is separated from EIF-1 by DEAE-cellulose chromatography.