Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two sets of experiments have been performed to test the DNA loop model of repression of the araBAD operon of Escherichia coli. First, dimethyl sulfate methylation protection measurements on normally growing cells show that the AraC regulatory protein occupies the araI site in the presence and absence of the inducer arabinose. Similarly, the araO2 site is shown to be occupied by AraC protein in the presence and absence of arabinose; however, its occupancy by AraC is greatly reduced when araI and adjacent sequences are deleted. Thus, AraC protein binds to araO2 cooperatively with some other component of the ara system located at least 60 base pairs away. Second, the mutational analysis presented here shows that the DNA components required for repression of araBAD are araI, araO2, and perhaps the araBAD operon RNA polymerase binding site.
...
PMID:The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites. 352 May 49

DNA primase (EC 2.7.7.6) produces an RNA oligomer of approximately 10 bases, which is required by DNA polymerase alpha (EC 2.7.7.7) for the initiation of DNA synthesis. We partially purified DNA primase from acute lymphocytic leukemia cells from patients using several chromatography columns. Poly(dT) and poly(dC), but not poly(dA) or poly(dG), were good templates for ribonucleoside triphosphate (rNTP)-dependent DNA synthesis (i.e., DNA primase activity), and they were used in the study of the effect of natural and arabinofuranosyl nucleoside triphosphates on DNA primase activity. The Km for GTP in the poly(dC) primase assay was approximately 175 microM. All noncomplementary natural rNTPs and deoxyribonucleoside triphosphates (dNTPs) inhibited poly(dC) primase activity to a similar extent (Ki values of ATP and CTP were 610 and 517 microM, respectively). 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) and 9-beta-D-arabinofuranosyladenine 5'-triphosphate (araATP) were more potent inhibitors of poly(dC) primase activity than were CTP and ATP (Ki values were approximately 125 microM). araCTP, araATP, CTP, and ATP inhibited DNA primase activity in a manner competitive with GTP. The concentration required to inhibit poly(dC) DNA primase activity by 50% was determined for a number of arabinofuranosyl nucleoside triphosphate analogs, and the relative potency of inhibition of DNA primase activity was as follows: rNTP = dNTP = 5-aza-dCTP less than ara-5-azaCTP = araTTP = araATP = araCTP less than 2-fluoro-araATP = 2'-azido-2'-deoxy araCTP less than 2'-fluoro-araTTP = 2'-fluoro-5-iodo-araCTP = 2'-fluoro-5-methyl-araCTP. In the poly(dT) primase assay ATP did not follow classic Michaelis-Menten kinetics (ATP exhibited positive cooperativity with a Hill coefficient of 2.0). However, this assay was very sensitive to araCTP (apparent Ki of 25 microM). In summary, these experiments suggested that DNA primase is controlled by the levels of ribonucleoside triphosphates, and that the perturbation of these pools by any agent could lead to the inhibition of DNA primase and thereby inhibit DNA synthesis. Furthermore, aranucleoside triphosphate analogs directly inhibited DNA primase, and it is possible that this effect may contribute to the cytotoxicity of these compounds.
...
PMID:Inhibition of DNA primase by nucleoside triphosphates and their arabinofuranosyl analogs. 380 92

The effects of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), both produced by E. coli, were measured on the activities of several genes in a cell-free system. Gene activity is measured as gene-directed synthesis of biochemically competent protein or transfer RNA. Both ppGpp and pppGpp stimulated the activities of the ara, lac, and trp operons and inhibited the arg operon. Production of transfer-RNA(Tyr) was unaffected by moderate levels of either ppGpp or pppGpp and only slightly inhibited at higher levels of ppGpp. Since the cell-free reaction mixtures hydrolyze pppGpp to ppGpp, we performed similar studies with a hydrolysis-resistant analog of pppGpp, the beta-gamma methylenyl derivative (pcppGpp). In general, pcppGpp shows the same inhibitory potency as pppGpp for the arg operon, but lacks the stimulatory effects on the ara, lac, and trp operons. This result suggests that the stimulation of these gene activities is specific for ppGpp.Under similar conditions, pppGpp and ppGpp show a slight inhibitory effect on the messenger-directed synthesis of beta-galactosidase and no effect on the messenger-directed synthesis of MS2 viral-coat protein. These observations, together with the fact that in the same system these nucleotides affect coupled transcription and translation, lead us to surmise that the activities of pppGpp and ppGpp are exerted at the level of RNA polymerase activity.
...
PMID:Effects of guanosine tetraphosphate, guanosine pentaphosphate, and beta-gamma methylenyl-guanosine pentaphosphate on gene expression of Escherichia coli in vitro. 435 31

The Escherichia coli lac and ara promoters and rrnC ribosomal RNA promoter-leader region were fused to lacZYA. Transcription termination signals were introduced into the lac genes of these fusions by Tn9 and IS1 insertions. Measurement of lac enzymes from upstream and downstream of the insertions showed that termination signals resulting from these insertions are very efficient when transcription begins at lac or ara promoters but are very inefficient when transcription begins at the rrnC promoter-leader region. The rrnC promoter-leader region must, therefore, modify RNA polymerase to enable it to read through transcription termination signals.
...
PMID:Antitermination of transcription from an Escherichia coli ribosomal RNA promoter. 620 58

The locations of DNA binding by the proteins involved with positive and negative regulation of transcription initiation of the L-arabinose operon in Escherichia coli have been determined by the DNase I protection method. Two cyclic AMP receptor protein sites were found, at positions -78 to -107 and -121 to -146, an araC protein--arabinose binding site was found at position -40 to -78, and an araC protein-fucose binding site was found at position -106 to -144. These locations, combined with in vivo data on induction of the two divergently oriented arabinose promoters, suggest the following regulatory mechanism: induction of the araBAD operon occurs when cyclic AMP receptor protein, araC protein, and RNA polymerase are all present and able to bind to DNA. Negative regulation is accomplished by the repressing form of araC protein binding to a site in the regulatory region such that it stimultaneously blocks access of cyclic AMP receptor protein to two sites on the DNA, one site of which serves each of the two promoters. Thus, from a single operator site, the negative regulator represses the two outwardly oriented ara promoters. This regulatory mechanism explains the known positive and negative regulatory properties of the ara promoters.
...
PMID:The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation. 625 57

The DNA-protein contact sites in the ara regulatory region, which contains the promoters for araBAD and araC, have been determined for araC protein, the cyclic AMP-binding protein, and RNA polymerase, by using the methylation protection and DNase I protection methods. The functional significance of binding was assessed by correlating the state of occupancy of these sites with promoter activity in transcription initiation. Our results suggest that the basis for araC autoregulation is that araC protein, in either its activator (P2) or repressor (P1) form, acts as a repressor for araC, by binding to the RNA polymerase attachment site at the araC promoter. We also found that the araC and araBAD promoters share a common site of positive control by the cyclic AMP-binding protein, located 90 bases from the araBAD and 60 bases from the araC transcriptional start points. A model for the mechanism of regulation of araBAD and araC expression by the catabolite gene-activator protein, P1, and Pe is proposed. An earlier model proposed by Ogden et al. [Ogden S., Haggerty, D., Stoner, C. M., Kolodrubetz, D. & Schleif, R. (1980) Proc. Natl. Acad. Sci, USA 77, 3346-3350] is discussed in the light of the data presented in this paper.
...
PMID:Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli. 626 69

Five mutations that result in reduced expression of the araBAD operon were cloned onto the plasmid pBR322. The position of each mutation was determined by DNA sequence analysis. Three of the mutations were located in the RNA polymerase binding site of the araBAD promoter. The first, ara-1016, was a one-base-pair deletion at position -35; the second, ara-1036, was a transversion at position -13; the third, ara-1027, was a nine-base-pair deletion from +5 to +13. S1 nuclease mapping showed that mutations ara-1016 and ara-1036 greatly reduced transcription and that mutation ara-1027 had little, if any, effect on transcription. Two other mutations resulted from the transposition of the insertion element, IS1, downstream from the transcriptional start site of the operon. Molecular mechanisms for all of the mutations are discussed.
...
PMID:Five mutations in the promoter region of the araBAD operon of Escherichia coli B/r. 631 19

9-beta-D-Arabinofuranosyl-6-mercaptopurine (ara-6-MP) was used to affinity-label wheat germ DNA-dependent RNA polymerase II (or B) (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). This nucleoside analogue was found to be a competitive inhibitor with respect to [3H]UMP incorporation. Natural substrates protected the enzyme from inactivation by ara-6-MP when the enzyme was preincubated with excess concentrations of substrates, suggesting that the inhibitor binds at the elongation subsite. The inhibitor bound the catalytic center of the enzyme with a stoichiometry of 0.6:1. The sulfhydryl reagent, dithiothreitol, reversed the inhibition by ara-6-MP, suggesting that the 6-thiol group of the inhibitor was interacting closely with an essential cysteine residue in the catalytic center of the enzyme. Chromatographic analysis of the pronase-digestion products of the RNA polymerase II-ara-6-MP complex also showed that ara-6-MP had bound a cysteine residue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured [6-35S]ara-6-MP-labeled RNA polymerase II revealed that over 80% of the radioactivity was associated with the IIb subunit of the enzyme.
...
PMID:Probes of eukaryotic DNA-dependent RNA polymerase II-I. Binding of 9-beta-D-arabinofuranosyl-6-mercaptopurine to the elongation subsite. 681 39

Phage P4 DNA is replicated in cell-free extracts of Escherichia coli in the presence of partially purified P4 alpha protein [Krevolin and Calendar (1985), J. Mol. Biol. 182, 507-517]. Using a modified in vitro replication assay, we have further characterized this process. Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated molecules demonstrates that the system yields supercoiled monomeric DNA as the main product. Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates in vitro mainly at ori, the origin of replication used in vivo. Replication proceeds from this origin bidirectionally, resulting in theta-type molecules. In contrast to the in vivo situation, no extensive single-stranded regions were found in these intermediates. The initiation proteins of the host, DnaB and DnaG, and the chaperones DnaJ and DnaK are not required for P4 replication, because polyclonal antibodies against those polypeptides do not inhibit the process. The reaction is inhibited by antibodies against the SSB protein, and by ara-CTP, a specific inhibitor of DNA polymerase III holoenzyme. Consistent with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase. Novobiocin, a DNA gyrase inhibitor, strongly inhibits P4 DNA synthesis, indicating that form I DNA is the required substrate.
...
PMID:Phage P4 DNA replication in vitro. 802 13

The effects of transient expression of poliovirus 2A(pro) on p220 cleavage in COS cells have been analyzed. When 2A(pro) was cloned in plasmid pTM1 and transiently expressed in COS cells, efficient cleavage of p220 occurred after infection of these cells with a recombinant vaccinia virus bearing phage T7 RNA polymerase. High numbers of COS cells were transfected with pTM1-2A, as judged by p220 cleavage, thereby allowing an analysis of the effects of poliovirus 2A(pro) on vaccinia virus gene expression. A 40-50% cleavage of p220 by transfected poliovirus 2A(pro) was observed ten hours post infection and cleavage was almost complete (80-90%) 20-25 hours post infection with vaccinia virus. Profound inhibition of vaccinia virus protein synthesis was detectable ten hours post infection and was maximal 20-25 hours post infection. This inhibition resulted from neither a blockade of transcription of vaccinia virus nor a lack of translatability of the mRNAs present in cells that synthesize poliovirus 2A(pro). Addition of ara-C inhibited the replication of vaccinia virus and allowed the continued synthesis of cellular proteins. Under these conditions, 2A(pro) is expressed and blocks cellular translation. Finally, p220 cleavage by 2A(pro) did not inhibit the translation of a mRNA encoding poliovirus protein 2C, as directed by the 5' leader sequences of encephalomiocarditis virus. Therefore, these findings show a correlation between p220 cleavage and inhibition of translation from newly made mRNAs. Our results are discussed in the light of present knowledge of p220 function, and new approaches are considered that might provide further insights into the function(s) of initiation factor eIF-4F.
...
PMID:Effects of poliovirus 2A(pro) on vaccinia virus gene expression. 857 44


<< Previous 1 2 3 Next >>

---