Search PubMed⌕ Search

Biomedical subjects

T Blumenthal

Publications and source records attributed to T Blumenthal.

At least 73 records · Page 4Linked to original sources
PubMed1980

Interaction of host-coded and virus-coded polypeptides in RNA phage replication.

The enzymes responsible for replication of the RNA of the single-stranded RNA bacteriophages contain, in addition to one phage-coded polypeptide, three host-coded polypeptides taken from the protein biosynthetic machinery: ribosomal protein S1 and the elongation factors Tu and Ts. While S1 performs a function in RNA replication derived from its protein synthetic function, mRNA binding, the reactions catalysed by the elongation factors in protein synthesis are apparently dispensible for RNA replication. In the replicase, these polypeptides, acting as the EF-Tu . Ts complex, play a fundamental structural role. Replacement of the endogenous EF-Tu with mutant EF-Tu, itself stable, causes the RNA replicase to become unstable. The possibility that EF-Tu . Ts is solely a structural protein in the RNA replicase is suggested by experiments showing that a variety of modifications of the elongation factors can be tolerated without loss of RNA synthetic capacity. In fact, EF-Tu . Ts from distantly related bacterial species can substitute for E. coli EF-Tu . Ts in RNA replicase. Evidence is presented that the high in vitro template specificity of Q beta replicase may be accomplished through modulation of the level of GTP required for initiation of transcription. Different natural and synthetic RNAs require quite different GTP concentrations. Mn2+ ions, which extend the range of templates transcribed by Q beta replicase, lower the requirement for GTP. High ionic strength, which alters the conformation of Q beta replicase such that template specificity is increased, raises the GTP requirement. An additional host coded protein required for in vitro Q beta RNA replication, host factor (HF), interacts specifically with Q beta RNA. This polypeptide acts by allowing Q beta replicase to initiate RNA synthesis with Q beta RNA at reduced GTP concentration.

Coliphages↗
PubMed1980

Q beta replicase containing wild type and mutant tufA and tufB gene.

The protein synthesis elongation factor EF-Tu, complexed with EF-Ts, forms part of Q beta RNA replicase. In an effort to determine its function in the RNA synthesis reaction, we have developed procedures which allow us to replace the endogenous EF-Tu in purified Q beta replicase with EF-Tu from a variety of sources. In this communication we report purification of EF-Tu from strains containing (a) a wild type tufA gene only, (b) a kirromycin-resistant mutant tufA gene only, and (c) a kirromycin-resistant mutant tufA gene and a mutant tufB gene which codes for EF-Tu that does not bind ribosomes. When each of these EF-Tu preparations is inserted in Q beta replicase, the wild type tufA gene product and and the tufB gene product function appearently normally, but the kirromycin-resistant tufA gene product causes the formation of an altered enzyme. The Q beta replicase containing kirromycin-resistant EF-Tu is unstable; it is rapidly inactivated in the reaction mixture, even at temperatures as low as 20 degrees C. This property results in an apparent increase in template specificity; while wild type Q beta replicase will transcribe poly(C) and other synthetic RNA species, the mutant enzyme will do so only in the presence of Mn2+, which reduces template specificity. The kirromycin-resistant Q beta replicase will also transcribe Q beta RNA. The results imply that EF-Tu is involved in maintenance of enzyme structure, which, in turn, is implicated in template specificity.

Anti-Bacterial Agents↗
PubMed1980

Q beta replicase template specificity: different templates require different GTP concentrations for initiation.

Qbeta replicase is notable for its high degree of template specificity. It has been shown to transcribe Qbeta RNA and synthetic polymers containing cytidylate. However, other natural RNAs are not transcribed unless Mn2+ ions are present. The enzyme initiates all RNA synthesis with GTP. In this report it is shown that Qbeta replicase can transcribe heterologous natural RNA species in the absence of Mn2+ if sufficient GTP is present. Each RNA tested requires a different GTP concentration for initiation. These results indicate that the site for the initiating nucleoside triphosphate on Qbeta replicase is strongly influenced by the template. It is proposed that the high degree of template specificity is a consequence of the fact that different templates induce initiation sites with varying affinities for GTP. Two lines of evidence support this idea. First, Mn2+ ions, which reduce template specificity, reduce the concentration of GTP required for initiation. Second, high ionic strength, which decreases transcription of all templates except Qbeta RNA, increases the GTP requirement. The possibility is considered that variable promoter or ribosome binding site strengths could result from a mechanism similar to that proposed here.

Coliphages↗
PubMed1980

Protein synthesis elongation factors Tu and Tu.Ts from Caulobacter crescentus: sensitivity to kirromycin and activity in Q beta replicase.

The protein synthesis elongation factors Tu and Ts are responsible for binding aminoacyl-transfer ribonucleic acid (RNA) to the ribosome. In addition, they perform an undefined function, as the EF-Tu.Ts complex, in the RNA phage RNA replicases. In an effort to obtain insight into these two apparently unrelated roles, we purified the elongation factors from Caulobacter crescentus and compared them to the analogous Escherichia coli polypeptides. Although most physical and functional characteristics were found to be similar, significant differences were found in the molecular weight of EF-Ts and relative affinities of guanine nucleotides, sensitivity to trypsin cleavage, and rate of heat denaturation of EF-Tu. The antibiotic kirromycin was active with EF-Tu from both bacterial species. When C. crescentus EF-Tu.Ts was substituted for the E. coli elongation factors in Q beta phage RNA replicase, an enzyme capable of apparently normal RNA synthetic activity was formed.

Anti-Bacterial Agents↗
PubMed1977

Host factor for coliphage Q beta RNA replication: presence in procaryotes and association with the 30S ribosomal subunit in Escherichia coli.

The Host Factor required for in vitro coliphage Q beta RNA replication, a heat-stable RNA binding protein present in uninfected Escherichia coli, has been detected by both immunological and functional tests in Acinetobacter calcoaceticus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Pseudomonas putida. It was not detectable by these criteria in Bacillus stearothermophilus, Bacillus subtilis, Caulobacter crescentus, Micrococcus lysodeikticus, Rhodopseudomonas capsulata or Saccharomyces cerevisiae. In Escherichia coli the Host Factor protein has been shown to be associated with ribosomes. It is demonstrated here that this association is specific for the 30S ribosomal subunit.

Acinetobacter↗
PubMed1977

Conformational alteration of protein synthesis elongation factor EF-Tu by EF-Ts and by kirromycin.

Alterations of the structure of EF-Tu have been investigated by using the rate of EF-Tu cleavage by trypsin as a conformational probe. The presence of EF-Ts bound to EF-Tu results in a 10-fold increase in the cleavage rate. The antibiotic kirromycin, which inhibits protein synthesis by virtue of its interaction with EF-Tu, mimics this effect of EF-Ts. Both kirromycin and EF-Ts also facilitate the exchange of free GDP with GDP bound to EF-Tu. The results suggest that EF-Ts and kirromycin induce a similar conformational change in EF-Tu, thereby "opening" the guanine nucleotide binding site. The trypsin-cleaved EF-Tu still can bind GDP and EF-Ts and can function in Qbeta replicase, but it no longer spontaneously renatures following denaturation in urea.

Anti-Bacterial Agents↗
PubMed1977

Cell-mediated immune response in gonococcal infections.

Peripheral blood lymphocyte (PBL) transformation stimulated by gonococcal and meningococcal antigens was studied in 29 men and 21 women with uncomplicated gonorrhoea. The blastogenic responses of PBLs from these men and women were substantially higher than from normal controls. Cross-reactivity between Neisseria gonorrhoeae and Neisseria meningitidis was manifested by the PBL transformation responses in patients with gonorrhoea to non-purified meningococcal antigen (MGC-I). In both male and female patients the PBLs were stimulated by non-purified gonococcal antigen (GC-I) and by non-purified meningococcal antigen. The extent of the blastogenic response in women was much greater than in men. Partial purification of these antigens by gel chromatography resulted in reduced cross-reactive responses to the semi-purified meningococcal antigen (MGC-II). Female patients demonstrated marked stimulation with the semi-purified gonococcal antigen (GC-II), while male patients showed slight stimulation with GC-II. It is possible that cell-mediated immunity may act to limit the spread of gonococcal infection beyond the genital mucous membranes.

Antigens, Bacterial↗
PubMed1976

Function and structure in ribonucleic acid phage Qbeta ribonucleic acid replicase. Effect of inhibitors of EF-Tu on ribonucleic acid synthesis and renaturation of active enzyme.

Escherichia coli Phage Qbeta RNA replicase, an RNA-dependent RNA polymerase, is a tetramer composed of one phage-coded polypeptide and three host-supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host. Two of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, are required for initiation of transcription by Qbeta replicase with all templates. Using a previously developed reconstitution system we have examined the effects of modification of EF-Tu on reconstituted replicase activity. The poly(G) polymerase activity of the enzyme can be recovered after pretreatment of the EF-Tu-GDP with either L-1-tosylamido-2-phenylethyl chloromethyl ketone or N-ethylmaleimide, both of which inhibit the aminoacyl-tRNA binding activity of EF-Tu. This suggests that the aminoacyl-tRNA binding site of EF-Tu is not required for Qbeta replicase activity. When Qbeta replicase is treated with kirromycin, an antibiotic which modifies EF-Tu activity by an unknown mechamism, the protein synthetic activity of the EF-Tu in the replicase complex is eliminated but the Qbeta RNA replication activity is only slightly affected. Treatment of pure EF-Tu with kirromycin, however, prevents it from functioning in the renaturation of Qbeta replicase. This antibiotic is not effective against the EF-Tu-Ts complex in the reconstitution assay. Kirromycin at the relatively high concentration used here is found to prevent the formation of the EF-Tu-Ts complex. GDP, which binds to EF-Tu and inhibits formation of the complex with EF-Ts, also inhibits renaturation of Qbeta replicase. It is suggested that the EF-Tu-Ts complex, rather than the individual polypeptides, functions in the renaturation of Qbeta replicase and that the kirromycin and GDP act by preventing formation of this complex.

Anti-Bacterial Agents↗
PubMed1976

Function and structure in phage Qbeta RNA replicase. Association of EF-Tu-Ts with the other enzyme subunits.

Qbeta replicase is a complex of four nonidentical subunits readily dissociable into two subcomplexes: 30 S ribosomal protein S1 and the phage-coded polypeptide (Subunits I + II) and protein synthesis elongation factors EF-Tu and EF-Ts (Subunits III + IV). The affinity of the two subcomplexes for one another increases with increasing ionic strength. The enzyme is capable of initiation of RNA synthesis with synthetic templates only when in the low ionic strength conformation. Elongation of initiated polynucleotide chains is not affectedby ionic strength. Addition of Qbeta RNA to the enzyme also alters its quaternary structure: the EF-Tu-Ts cannot be covalently attached to the other enzyme subunits with bifunctional cross-linking reagents in the presence of RNA. This conformational change is not influenced by ionic strength. The addition of Qbeta RNA to the enzyme, does not result in the release of EF-Tu-Ts from the other enzyme subunits: whereas free EF-Tu-Ts binds GDP independently of salt concentration, this binding by Qbeta replicase is sensitive to high ionic strength and remains so in the presence of Qbeta RNA. Furthermore, RNA does not allow the release of EF-Ts from EF-Tu by GTP as measured by sensitivity of EF-Ts activity to N-ethylmaleimide.

Binding Sites↗
PubMed1976

Renaturation of a multisubunit multiactivity enzyme complex: recovery of phage Qbeta RNA replicase, EF-Tu, and EF-Ts activities after denaturation in urea.

Phage Qbeta RNA replicase consists of four nonidentical subunits three of which are required for poly(C)-directed synthesis of poly(G): a phage-coded polypeptide and the two host-supplied protein biosynthesis elongation factors EF-Tu and EF-Ts. After denaturation of the enzyme in 8 M urea, poly(G) polymerase activity can be renaturated by dilution of the denatured subunits into a high ionic strength buffer with glycerol. The renaturation reaction has a broad temperature optimum between 11 and 21 degrees. The extent of renaturation is dependent on enzyme concentration: at low enzyme concentrations and 21 degrees renaturation proceeds for more than 3 h with greater than 40% recovery of activity, whereas at high enzyme concentrations the reaction is complete by 1 h with less than 10% of the poly(G) polymerase activity regained. Activities catalyzed by the elongation factors can be measured while they are part of the replicase complex. Study of rates of renaturation of EF-Tu and EF-Ts dependent activities alone and in the replicase complex revealed that virtually 100% of the EF-Ts activity was recovered more rapidly than could be assayed at temperatures as low as 2 degrees, while the rate of recovery of EF-Tu activity was comparable to that of the poly(G) polymerase activity and was independent of either EF-Tu concentration or the presence of other enzyme subunits. The rate of recovery of the poly(G) polymerase activity was found to be limited by the renaturation of EF-Tu, since the rate was dramatically increased by the addition of undenatured EF-Tu.

Binding Sites↗
PubMed1976

Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex.

Escherichia coli phage Qbeta RNA replicase, an RNA-dependent RNA polymerase (RNA-dependent RNA nucleotidyltransferase), is a tetramer composed of one phage-coded polypeptide and three host-supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host. Two of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, can be covalently crosslinked with dimethyl suberimidate to form a complex which lacks the ability to catalyze the known host functions catalyzed by the individual elongation factors. Using a previously developed reconstitution system we have examined the effects of crosslinking the EF-Tu-Ts complex on reconstituted replicase activity. Renaturation is significantly more efficient when exogenously added native EF-Tu-Ts is crosslinked than when it is not. Crosslinked EF-Tu-Ts can be purified from a crude crosslinked postribosomal supernatant by its ability to replace EF-Tu and EF-Ts in the renaturation of denatured Qbeta replicase. A sample of Qbeta replicase with crosslinked EF-Tu-Ts replacing the individual elongation factors was prepared. Although it lacked EF-Tu and EF-Ts activities, it could initiate transcription of both poly(C) and Qbeta RNA normally and had approximately the same specific activity as control enzyme. Denatured Qbeta replicase formed with crosslinked EF-Tu-Ts was found to renature much more rapidly than untreated enzyme and, in contrast to normal replicase, its renaturation was not inhibited by GDP. The results demonstrate that EF-Tu and EF-Ts function as complex in Qbeta replicase and do not perform their known protein biosynthetic function in the RNA synthetic reaction.

Chemical Phenomena↗
PubMed1975

Host factor for coliphage Qbeta RNA replication is present in Pseudomonas putida.

Host Factor (HF)1, is a 12000 molecular weight polypeptide that is found in uninfected Escherichia coli and is required as a hexamer along with Qbeta replicase for in vitro replication of Qbeta phage RNA. It has recently been found to be associated with ribosomes and to bind tightly to poly(A). We report here the identification and purification of HF from Pseudomonas putida. HF can be detected in crude extracts by both functional activity in the Qbeta RNA replication assay and by immunodiffusion with antibody made against E. coli HF. HF from E. coli and P. putida chromatograph similarly on DEAE-cellulose and phosphocellulose. They have similar but not identical molecular weights as judged by SES-polyacrylamide gel electrophoresis. Like E. coli HF, P. putida HF was found to be associated with ribosomes and to bind tightly to poly(A). Furthermore, the pure protein from P. putida has full funcitonal activity in the in vitro Qbeta RNA replication assay. The findings that HF has been conserved during evolution, is associated with ribosomes, and binds poly(A), suggest that HF may be an important translational element in uninfected cells and that its role involves an interaction with RNA.

Coliphages↗