Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

(1) The RNA replicase induced by bacteriophage Qbeta consists of four non-identical subunits designated as alpha (mol. wt. 74000), beta (mol. wt. 64000), gamma (mol. wt. 47000) and delta (mol. wt. 33000), only one (subunit beta) of which is specified by the phage genome. (2) Subunit alpha (30 S ribosomal protein "S1" as well as translational interference factor "i") is required only for (+) strand-directed RNA synthesis in the presence of the host factor. (3) Qbeta replicase lacking subunit alpha (R-alpha) is capable of replicating templates other than (+) strand, such as (--), "6S" RNA, poly(C) etc., in the absence of the host factor. (4) Subunit beta is suggested to be the nucleotide-polymerizing enzyme, but is unable to initiate RNA synthesis by itself. (5) Subunits gamma and delta are identical to the protein synthesis elongation factors, EF-Tu and EF-Ts, respectively, and are required only for initiation of RNA synthesis, but not for elongation. (6) A model of Qbeta replicase is presented in order to discuss observed template-enzyme interactions.
 ...
PMID:Structure and function of RNA replicase of bacteriophage Qbeta. 5 11

Phage SP RNA-dependent RNA polymerase (SP replicase) was purified from Escherichia coli infected with RNA phage SP. The enzyme was found to be composed of four non-identical polypeptides, i.e. subunits I, II, III, and IV and molecular weights of 74,000, 69,000, 47,000, and 36,000 daltons, respectively. As in the case of phage Qbeta replicase, the largest polypeptide is identical with the ribosomal protein S1, and subunits III and IV with polypeptide chain elongation factors EF-Tu and EF-ts, respectively.. This is based on the behaviour of the subunits on SDS-polyacrylamide gel electrophoresis, isoelectric focusing and immunological cross-reaction. Subunits I, III, and IV of SP replicase are derived from the host cell, while subunit II is coded by phage RNA genome. The striking coincidence of the composition and entity of the structural components of SP replicase with those of Qbeta replicase may indicate the structural and functional requirements of host-derived polypeptides in RNA replicase. The binding activity of S1 (in 70S ribosome comples) to poly (U) is retained in SP replicase complex. In contrast, the GDP binding activity of EF-Tu is masked in SP replicase. It is concluded that S1 is required functionally whereas EF-Tu.EF-Ts are required structurally in RNA replicase.
 ...
PMID:Identification of host-derived subunits of phage SP RNA-dependent RNA polymerase (SP replicase). 36 4

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.
 ...
PMID:Renaturation of a multisubunit multiactivity enzyme complex: recovery of phage Qbeta RNA replicase, EF-Tu, and EF-Ts activities after denaturation in urea. 76 66

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.
 ...
PMID:Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex. 106 92

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.
 ...
PMID: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. 126 42

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.
 ...
PMID:Protein synthesis elongation factors Tu and Tu.Ts from Caulobacter crescentus: sensitivity to kirromycin and activity in Q beta replicase. 610 49

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.
 ...
PMID:Q beta replicase containing wild type and mutant tufA and tufB gene. 698 24

In researches involving in vitro protein synthesis and self-replication system, Qbeta replicase is one of the key enzymes, which are demanded for the high availability. Qbeta replicase is a RNA-dependent RNA polymerase of Qbeta coliphage. It consists of four subunits (alpha, beta, gamma, and delta subunit), where the beta-subunit is encoded by the viral genome, while the other three subunits are host proteins normally involved in protein synthesis, namely, ribosomal protein S1 (alpha), elongation factors EF-Tu (gamma) and EF-Ts (delta). To increase the production of the Qbeta replicase holoenzyme, several types of expression vectors, including pKK, pET and others, were employed to produce Qbeta replicase. However, the beta-subunit was almost in the precipitate fraction. Considering that the four subunits of Qbeta replicase holoenzyme are in equivalent molar ratio and the amount of the subunits, ribosomal S1 and EF-Ts, being produced by the host cells is relatively low, co-expression of beta-subunit with the other three subunits was performed to know whether the availability of the host subunits is the contributing factor for the solubility of the Qbeta replicase. pBAD33-rep was constructed by cloning the beta-subunit gene into pBAD 33, a pACYC derivative, and pET21a(+) was employed as expression vector for the three other subunits. Among the different combinations of co-expression experiments, solubility was found to slightly increase by SDS-PAGE analysis when the beta-subunit was co-expressed with EF-Tu-Ts. And the replicase activity assay showed this soluble enzyme is in active form. The expression of beta-subunit was enhanced by decreasing the level of inducer IPTG in co-expression, and more soluble enzyme were obtained.
 ...
PMID:[Co-expression of beta-subunit with other subunits of Qbeta replicase]. 1611 Sep 60

Qbeta replicase functioning in Escherichia coli is an RNA-dependent RNA polymerase composed of one phage-coded subunit and three host-coded proteins: ribosomal protein S1, and protein elongation factors EF-Tu and EF-Ts. Qbeta replicase lacking ribosomal protein S1 (alpha-less replicase) is capable of replicating some small RNAs. We attempted to create functional alpha-less replicase by co-expression of the mRNAs that code for the subunits of alpha-less replicase in a rabbit reticulocyte cell-free translation system. Replicase activity, however, could not be detected when both EF-Tu and EF-Ts were co-expressed with the phage-coded subunit. On the other hand, active alpha-less replicase was obtained when an EF-Ts-EF-Tu fusion protein was co-expressed with the phage-coded subunit. Consequently, we succeeded in generating genetically engineered active alpha-less Qbeta replicase which functions in a eukaryotic cell-free system.
 ...
PMID:Genetically engineered active Qbeta replicase in rabbit reticulocyte cell-free system: a fusion protein of EF-Tu and EF-Ts is functional as the subunit of Qbeta replicase. 1623 59

Qbeta replicase, an RNA-dependent RNA polymerase of RNA coliphage Qbeta, is a heterotetramer composed of a phage-encoded beta-subunit and three host-encoded proteins: the ribosomal protein S1 (alpha-subunit), EF-Tu, and EF-Ts. Several purification methods for Qbeta replicase were described previously. However, in our efforts to improve the production of Qbeta replicase, a substantial amount of the beta-subunit overproduced in Escherichia coli cells was found as insoluble aggregates. In this paper, we describe two kinds of method of producing Qbeta replicase. In one kind, both EF-Tu and EF-Ts subunits were expressed with the beta-subunit, and in the other kind, the beta-subunit was genetically fused with EF-Tu and EF-Ts. The fused protein, a single-chain alpha-less Qbeta replicase, was mostly found in the soluble fraction and could be readily purified. These results pave the way for the large-scale production of the highly purified form of this enzyme.
 ...
PMID:Functional Qbeta replicase genetically fusing essential subunits EF-Ts and EF-Tu with beta-subunit. 1678 72


1 2 Next >>