PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Peptide Chain Elongation, Translational”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1998

Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex.

RF3 was initially characterized as a factor that stimulates translational termination in an in vitro assay. The factor has a GTP binding site and shows sequence similarity to elongation factors EF-Tu and EF-G. Paradoxically, addition of GTP abolishes RF3 stimulation in the classical termination assay, using stop triplets. We here show GTP hydrolysis, which is only dependent on the simultaneous presence of RF3 and ribosomes. Applying a new termination assay, which uses a minimessenger RNA instead of separate triplets, we show that GTP in the presence of RF3 stimulates termination at rate-limiting concentrations of RF1. We show that RF3 can substitute for EF-G in RRF-dependent ribosome recycling reactions in vitro. This activity is GTP-dependent. In addition, excess RF3 and RRF in the presence of GTP caused release of nonhydrolyzed fmet-tRNA. This supports previous genetic experiments, showing that RF3 might be involved in ribosomal drop off of peptidyl-tRNA. In contrast to GTP involvement of the above reactions, stimulation of termination with RF2 by RF3 was independent of the presence of GTP. This is consistent with previous studies, indicating that RF3 enhances the affinity of RF2 for the termination complex without GTP hydrolysis. Based on our results, we propose a model of how RF3 might function in translational termination and ribosome recycling.

GTP Phosphohydrolase-Linked Elongation Factors↗
PubMed1994

Rit1, a tRNA backbone-modifying enzyme that mediates initiator and elongator tRNA discrimination.

Using a genetic screen in yeast aimed at identifying cellular factors involved in initiator and elongator methionine tRNA discrimination in the translational process, we have identified a mutation that abolish the requirement for elongator methionine tRNA. The gene affected, which we call the ribosylation of the initiator tRNA gene or RIT1, encodes a 2'-O-ribosyl phosphate transferase. This enzyme modifies exclusively the initiator tRNA in position 64 using 5'-phosphoribosyl-1'-pyrophosphate as the modification donor. As the initiator tRNA participates both in the initiation and elongation of translation in a rit1 strain, we conclude that the 2'-O-ribosyl phosphate modification discriminates the initiator tRNAs from the elongator tRNAs during protein synthesis. The modification enzyme was shown to recognize the stem-loop IV region that is unique in eukaryotic cytoplasmic initiator tRNAs.

Alleles↗
PubMed2000

Late events of translation initiation in bacteria: a kinetic analysis.

Binding of the 50S ribosomal subunit to the 30S initiation complex and the subsequent transition from the initiation to the elongation phase up to the synthesis of the first peptide bond represent crucial steps in the translation pathway. The reactions that characterize these transitions were analyzed by quench-flow and fluorescence stopped-flow kinetic techniques. IF2-dependent GTP hydrolysis was fast (30/s) followed by slow P(i) release from the complex (1.5/s). The latter step was rate limiting for subsequent A-site binding of EF-Tu small middle dotGTP small middle dotPhe-tRNA(Phe) ternary complex. Most of the elemental rate constants of A-site binding were similar to those measured on poly(U), with the notable exception of the formation of the first peptide bond which occurred at a rate of 0.2/s. Omission of GTP or its replacement with GDP had no effect, indicating that neither the adjustment of fMet-tRNA(fMet) in the P site nor the release of IF2 from the ribosome required GTP hydrolysis.

Codon↗
PubMed1993

The yeast initiator tRNAMet can act as an elongator tRNA(Met) in vivo.

Saccharomyces cerevisiae uses two different methionine accepting tRNAs during protein synthesis. One, tRNA(iMet), is used exclusively during the initiation of translation whereas the other, tRNA(mMet), is used during the elongation of translation. To study the unique features of each methionine tRNA species, we constructed yeast strains with null alleles of the five elongator methionine tRNA (EMT) genes and strains with null alleles of the four initiator methionine tRNA (IMT) genes, respectively. Consequently, growth of these strains was dependent either on a tRNA(mMet) or a tRNA(iMet), respectively, encoded from a plasmid-derived gene. For both null mutants, the plasmid carrying the wild-type gene can be selected against and exchanged for another plasmid derived EMT or IMT gene (wild-type or mutant). A high gene dosage of the wild-type IMT gene could restore growth to the elongator-depleted strain. However, wild-type EMT genes in a high gene dosage never restored growth of the initiator depleted strain. Thus, the elongator tRNA(Met) is much more restricted to participate in the initiation of translation than the initiator tRNA(Met) is restricted to participate in the elongation process. Using the two null mutants, we have identified tRNA(mMet) mutants, which show reduced elongator activity, and tRNA(iMet) mutants, with improved elongator activity in the elongator depleted strain. Also, tRNA(mMet) mutants that function as an initiator tRNA in the initiator depleted strain were identified. From this mutant analysis, we showed that the conserved U/rT at position 54 of the elongator tRNA(Met) is an important determinant for an elongator tRNA. The most important determinant for an initiator was shown to be the acceptor stem and especially the conserved A1.U72 base-pair. Mutant tRNAs, with reduced activity in either process, were investigated for enhanced activity during overproduction of the alpha and beta-subunits of the eukaryotic initiation factor 2 (eIF-2) or the eukaryotic elongation factor 1 alpha (eEF-1 alpha). The data suggest that the U/rT of the elongator at position 54 is important for eEF-1 alpha recognition and that the acceptor stem of the initiator is important for eIF-2 recognition.

Base Sequence↗
PubMed1997

Regulation of the elongation-termination decision at intrinsic terminators by antitermination protein N of phage lambda.

The mechanisms that control N-protein-dependent antitermination in the phage lambda life cycle have counterparts in the regulatory systems of other organisms. Here we examine N-dependent antitermination at the intrinsic tR' terminator of lambda to elucidate the regulatory principles involved. The tR' terminator consists of a sequence of six base-pairs along the template at which the transcription complex is sufficiently destabilized to make RNA release possible. Within this "zone of opportunity" for termination the termination efficiency (TE) at each template position is determined by a kinetic competition between alternative reaction pathways that lead either to elongation or to termination. TE values at each position within tR' have been mapped as a function of NTP concentration, and it is shown that N protein (in the presence of NusA and a nut site; the minimal system for N-dependent antitermination) can offset increases in TE that are induced by limiting the concentrations of each of the next required NTPs. By limiting NTP concentrations or working at low temperature we show that a significant effect of N within the minimal system is to increase the rate of transcript elongation three- to fivefold at most positions along the template. Assuming that a comparable increase in elongation rate applies at template positions within the terminator, we show that an increase of this magnitude is not sufficient to account for the antitermination efficiency observed and that an approximately 100-fold stabilization of the transcription complex at intrinsic termination sites as a consequence of binding the N-containing antitermination sub-assembly must be invoked as well. A general method for partitioning TE effects in antitermination between changes in elongation rate and termination complex stability is demonstrated, based on competing free energy of activation barriers for the elongation and termination reactions. The analysis and utility of such mixed modes of transcriptional regulation are considered in general terms.

Bacterial Proteins↗
PubMed1998

Combinatorial effects of NusA and NusG on transcription elongation and Rho-dependent termination in Escherichia coli.

The transcription factors NusA and NusG from Escherichia coli are modulators of the RNA polymerase elongation reaction and Rho-dependent transcription termination. NusA decreases the elongation rate and termination efficiency while NusG increases both activities. Both Nus factors are able to physically interact with Rho and with RNA polymerase. Experiments with purified components designed to determine whether these factors act independently or competitively showed that the change in elongation rate was a composite of their individual effects, that the combined effect on termination was dependent on the reaction conditions and that the two factors do not compete for their sites of action for either effect. The two factors were also found not to enhance significantly the slight (20%) inhibition of elongation caused by 200 microM guanosine 3',5'-bisdiphosphate (ppGpp) during transcription in vitro. The results also show that the effects of NusA and NusG on RNA polymerase elongation and Rho function are contrary to the inverse relationship between elongation and termination that is expected for a kinetic coupling of Rho action to RNA polymerase elongation. This property suggests that in addition to their known actions on RNA polymerase that influence the length of pausing, these factors act on some other rate-limiting step of the Rho-dependent termination process.

Bacterial Proteins↗
PubMed1991

A proposed role for IF-3 and EF-T in maintaining the specificity of prokaryotic initiation complex formation.

Initiation factor-free 30S subunits of E. coli ribosomes bind aminoacyl-tRNAs more efficiently than fMet-tRNA(fMet). Elongator-tRNA binding was unaffected by IF-1 or IF-2 but was inhibited by IF-3. Their combination reduced this binding up to 40% and stimulated that of fMet-tRNA(fMet). Unexpectedly, EF-T also prevented elongator-tRNA binding by complexing both to the 30S and to the aminoacyl-tRNAs. Using AUGU3 as mRNA, elongator-tRNAs competed with fMet-fRNA(fMet) and with tRNA(fMet), fMet-tRNA(fMet) reacted with puromycin after addition of 50S subunits suggesting that it occupied the P site. EF-T directed binding of phe-tRNA to the 30S.AUGU3 complex at the A site only if fMet-tRNA(fMet) or tRNA(fMet) filled the P/E site. We propose that one function of EF-T may be to prevent the entry of aminoacyl-tRNAs into the 30S particle during initiation. The possibility that a special site for fMet-tRNA resides on 16S rRNA is also discussed.

Binding Sites↗
PubMed1975

Activities of guanosine triphosphate analogues in reactions catalyzed by elongation factor Tu and initiation factor 2 of Escherichia coli.

In earlier studies two natural analogues of GTP, guanosine 3'-diphosphate 5'-triphosphate (pppGpp) and dGTP, were found to substitute for GTP in reactions catalyzed by initiation factor 2 (IF-2) and elongation factor Tu (EF-Tu), while only dGTP could replace GTP with elongation factor G. These observations with IF-2 and EF-Tu have been extended to two analogues of GTP modified at the 3' ribose hydroxyl position, 3'-deoxyguanosine 5'-triphosphate (3'dGTP) and 3'-deoxy-3'-aminoguanosine 5'-triphosphate (3'dNH2GTP). These compounds were found to be similar to GTP, dGTP, and pppGpp in IF-2-dependent formation of N-formylmethionyl-puromycin and EF-Tu-dependent formation of N-acetyl-Phe-Phe-tRNA. The apparent Km values for the five guanosine nucleotides were 2 - 10(-6)-4 - 10(-6)M in the former reaction and 2-10(-7)--6-10(-7) M in the latter. These reactions did not have an absolute requirement for either an intact pentose ring or for the guanine base in the nucleotide. Although substantially less active than the guanine nucleotides, ITP and the dialcohol derived from GTP by periodate oxidation and borohydride reduction (ox-redGTP) were partially active in both the IF-2 and EF-Tu-dependent reactions, with apparent Km values about 40-100 times those of GTP.

Escherichia coli↗
PubMed1976

Preferential regulation of protein synthesis initiation complex formation by purine nucleotides.

A comparison of the affinities of eukaryotic initiation factor 2 and eukaryotic elongation factor 1 for GTP and GDP, and of the responses of initiation and elongation complex formation to various GTP mol fractions indicated that the initiation reaction was more sensitive to changes in the GTP: GDP ratio. In vitro regulation of the GTP: GDP ratio by the adenylate energy charge, a sensitive control parameter, also demonstrated a preference for regulation of formation of initiation complexes when compared to elongation complexes. These studies suggest that, based on the availability of energy, initiation is the rate-limiting step in the overall protein synthetic process.

Animals↗