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

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

The effect of RNA secondary structure on rho-independent and rho-dependent termination of transcription of T3 DNA by Escherichia coli RNA polymerase has been studied by incorporating, into nascent transcripts, base analogs that lead to altered base-pairing properties. A guanine --> hypoxanthine substitution, with attendant weakening of secondary structure, abolished the rho-independent termination at 20% of the genome; in contrast, replacement of cytosine with 5-bromocytosine, which forms stronger pairs with guanine, enhanced termination at this site. rho-Independent termination was not altered by replacing uracil with 5-bromouracil. There are two major rho-dependent termination sites on the T3 DNA-at 8 and 15%. The termination activity of rho in this system also depended on RNA secondary structure. The incorporation of 5-bromouracil instead of uracil into RNA did not alter the site specificity of rho action but rho was rendered inactive when cytosine was replaced by 5-bromocytosine. In contrast, replacement of GTP with ITP in the reaction increased rho-dependent inhibition of RNA synthesis, caused production of heterogeneous-sized transcripts, and stimulated rho-mediated ATP hydrolysis. The rho-associated ATPase activity, in the presence of isolated T3 RNA, was also stimulated by inosine substitution. Furthermore, the temperature-sensitive rho isolated from rho 15 mutant of E. coli, which does not terminate transcription in the presence of the common rNTPs, was active when GTP was replaced with ITP. These results suggest that strongly paired G.C-rich regions in RNA stem-loop structures or RNA.DNA hybrids are essential for rho-independent termination, whereas rho-dependent termination requires weakly paired cytosine residues for its action.
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PMID:Termination of transcription by Escherichia coli RNA polymerase: influence of secondary structure of RNA transcripts on rho-independent and rho-dependent termination. 15 60

The study of transcription kinetics by T7 RNA polymerase is facilitated by the small size of its promoter, allowing the use of synthetic oligonucleotide templates with carefully defined sequences. We have previously used this approach to measure Michaelis-Menten steady-state kinetics for production of the five-base runoff transcript GGACU. In particular, Km for the interaction between enzyme and template under saturating levels of all four nucleotide triphosphates was shown to be approximately 0.02 microM. We now show that the corresponding Km and Vmax for initiation on a similar template coding for the runoff transcript GACU are the same as for the earlier study (Km = 0.02 microM; kcat = 40-50 min-1). This new template allows the measurement Km for association of the initial nucleotide GTP with enzyme or with the enzyme-DNA complex. The results show that KGTPm (0.60 mM) is somewhat higher than earlier approximations of Km for addition of elongating GTP during the later phase of processive elongation. As expected, the (initiating) Km for the GTP analogue ITP (KITPm) is increased (by about 2-fold), presumably as a result of weakened Watson-Crick base pairing. However, comparison of Km values for the GTP analogues GMP and guanosine shows little effect on substitution of the 5'-triphosphate by monophosphate or by a hydroxyl, respectively. This result suggests that a single active site has been evolutionarily adapted to accept from the 5' end of a waiting nucleotide both a 5'-triphosphate at initiation and a 5'-monophosphate ester (RNA) during elongation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:T7 RNA polymerase does not interact with the 5'-phosphate of the initiating nucleotide. 266 58

Transcription termination in vitro by vaccinia RNA polymerase is dependent on a trans-acting factor, VTF, that is associated with, if not identical to, the vaccinia mRNA capping enzyme. VTF-induced termination occurs approximately 50 nucleotides downstream of a signal sequence TTTTTNT in the non-transcribed templated strand; thus the cognate sequence UUUUUNU is expressed in the nascent RNA. To address the role of the nascent RNA in chain termination, the effects of nucleotide base analog substitutions were studied. Incorporation of bromo- (Br) UMP or iodo- (I) UMP into RNA abrogated factor-dependent termination without preventing the synthesis of read-through transcripts. Substitution of either ITP or 7'-methylguanosine for GTP did not inhibit factor-dependent termination, nor did the substitution of BrCTP or ICTP for CTP. The early transcripts synthesized in vitro were sensitive to RNase T2 but resistant to RNase H, indicating an absence of extensive hybridization of RNA product to the DNA template. Substitution of BrUTP for UTP did not alter the nuclease sensitivity of the transcripts, suggesting that increased stability of RNA:DNA hybrid structures did not account for the analog effects. These results are consistent with a model in which recognition of the primary sequence UUUUUNU in nascent RNA by the polymerase and/or VTF is required for transcription termination.
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PMID:Factor-dependent transcription termination by vaccinia virus RNA polymerase. Evidence that the cis-acting termination signal is in nascent RNA. 283 68

Premature termination of transcription has been demonstrated by eukaryotic RNA polymerase II at specific sites in the major late transcriptional unit of SV40 and in one of the transcriptional units of the parvovirus, minute virus of mice (MVM) (Y. Aloni and N. Hay, CRC Critical Reviews of Biochem., 18:327-383, 1985). In both cases the prematurely terminated (attenuated) RNA can be folded into a hairpin structure followed by U-residues that resemble a termination signal in prokaryotes. The experiments presented herein demonstrate premature termination of transcription 185 nucleotides (nt) downstream from the major late promoter of adenovirus type 2 (Ad2) in vivo, and in vitro in isolated nuclei and in HeLa whole cell extract. As in SV40 and MVM the attenuated RNA of Ad2 can be folded into a hairpin structure followed by U-residues. Transcription-termination was significantly reduced when ITP replaced GTP and when Br-UTP replaced UTP in the transcription reaction mixture, indicating that RNA secondary structure and the rU-dA interactions, respectively, are parts of the termination signal. Moreover, in isolated nuclei transcription-termination at the attenuation site occurred when the reaction mixture contained between 50-150 mM NaCl but not when it contained 300 mM NaCl. These results indicate that, at least in isolated nuclei, attenuation can be regulated. The possible involvement of termination factor(s) in the regulation of attenuation is discussed.
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PMID:Human RNA polymerase II can prematurely terminate transcription of the adenovirus type 2 late transcription unit at a precise site that resembles a prokaryotic termination signal. 350 41

We have selected from a mixture of random polynucleotides an RNA that is able to replicate in the presence of the DNA-dependent RNA polymerase of Escherichia coli, provided the medium contains Mn(2+) and ITP, in addition to ATP, CTP, and UTP. The RNA consists of a chain of about 125 (+/-25) nucleotides and appears to have a defined, nonrepetitive sequence.
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PMID:An RNA that multiplies indefinitely with DNA-dependent RNA polymerase: selection from a random copolymer. 457 40

Nuclei which were isolated from SV40 infected cells with a hypotonic detergent-free buffer were used to establish in vitro conditions which lead to transcription-termination at the attenuation site of SV40. This system allowed us to identify regulatory elements involved in transcription-termination by RNA polymerase B transcribing SV40. Transcription-termination at the attenuation site was found to be ionic strength dependent. Efficient termination occurred at low (100 mM NaCl) but not at high (100 mM (NH4)2 SO4 or 300 mM NaCl) ionic strength. When nuclei were prewashed with 300 mM NaCl, the efficiency of transcription-termination was low even when transcription was carried out at low ionic strength (100 mM NaCl). Efficient transcription-termination in the high salt prewashed nuclei was reconstituted by complementation with a high salt (300 mM NaCl) soluble factor extracted from nuclei of uninfected cells. In addition, the efficiency of transcription-termination was significantly reduced when ITP replaced GTP in the transcription reaction mixture. Our data indicate that a nuclear factor and RNA secondary structure are essential regulatory elements involved in transcription-termination by RNA polymerase B.
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PMID:Attenuation in SV40 as a mechanism of transcription-termination by RNA polymerase B. 632 6

6-Mercaptopurine was found to inhibit the growth of cultured human lymphoma P3HR-1 cells and the incorporation of [3H]-uridine into trichloroacetic acid-precipitable materials of the cells. One of the derivatives of 6-mercaptopurine, 6-mercaptopurine ribonucleoside triphosphate (6-thio-ITP), was found to inhibit in vitro RNA synthesis (both engaged and free enzyme activities) of the isolated nuclei from P3HR-1 cells. The alpha-amanitin-resistant RNA polymerase (polymerase I) and alpha-amanitin-sensitive RNA polymerase (polymerase II) of the cells were isolated and partially purified by either diethylaminoethyl cellulose or diethylaminoethyl Sephadex column chromatography, followed by DNA-cellulose affinity chromatography. It was found that these partially purified enzymes were also sensitive to 6-thio-ITP inhibition. Kinetic studies showed that the inhibition of RNA polymerase activities by 6-thio-ITP could be reversed by increasing concentrations of guanosine 5'-triphosphate in the reaction mixture, indicating that 6-thio-ITP may act as a competitive inhibitor of the enzymes by competing with guanosine 5'-triphosphate for its enzyme-binding site. These data suggest that inhibition of RNA transcription by 6-thio-ITP may be considered as one of the mechanisms of the cytotoxic action of 6-mercaptopurine in human tumor cells.
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PMID:Inhibition of human lymphoma DNA-dependent RNA polymerase activity by 6-mercaptopurine ribonucleoside triphosphate. 668 25

We have studied elongation of SV40 DNA F1 by E. coli RNA polymerase looking specifically at the length of the transcript as a function of time. By running the transcription reactions at 18 degrees C with limited enzyme and adding heparin or rifampicin after elongation has started, we have achieved almost exclusive initiation from the SV40 DNA preferred promotor size [Zain, B. S., Weissmann, S. M., Lebowitz, P., & Lewis, A. M., Jr. (1973) J. Virol. 11, 682-693]. In the region within 1500 nucleotides of the initiation we observe nine prominent sites and a number of minor site where hesitation during elongation occurs. The positions of these hesitation points or pause sites are not effected by changes in the salt concentration, the simultaneous lowering of the concentrations of all the NTPs, or by increases in the RNA polymerase concentration, implying that the pause sites are a consequence of the RNA, DNA, and RNA polymerase ternary complex. The pause sites are not an artifact of the lowered temperature (18 degrees C) used in the experiments since they are also observed at 37 degrees C. The first four of these sites have been sequenced by using the 3'-O-methyl analogues of the ribonucleotide triphosphates. We have found no sequence homology between the pause sites. The kinetics of the pause reactions do not fit a first-order model but do correspond to a scheme were continuation through a pause site and termination at a pause site are both represented. For one of the pause sites, the relaxation time for continuation through the pause site was determined to be approximately 2.5 min and for the termination approximately 50 min at 18 degrees C. If the concentration of one of the NTPs is lowered to 10 muM, the strength of a pause site can be increased if that NTP is contained in the pause. Also, minor pause sites are observed at regions in the RNA sequence which are rich in the NTP that has the lowered concentration. When GTP is replaced by ITP during transcription, a new set of pause site quite different from the normal sites of hesitation are observed. The major new pause sites occur at or near sequences in the RNA which are rich in I-U residues preceded by a region rich in C residues. This indicates, as has been previously noted, that sequences where the DNA.RNA hybrid is quite stable followed by a region that is very unstable may cause termination. When BrUTP replaced UTP, very little effect was observed on the pause sites. The addition of p termination factor causes termintion to increase in all the pause sites with a length greater than 300 nucleotides. In the type of experiments performed here, those pause sites had continuation relaxation times greater than 45 s at 37 degrees C. This implies that regardless of the nature of a pause, p will cause at least some termination at all hesitation sites with a relaxation time greater than 45 s. All the results are discussed in terms of a kinetic model for the termination of elongation.
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PMID:Escherichia coli deoxyribonucleic acid dependent ribonucleic acid polymerase transcriptional pause sites on SV40 DNA F1. 701 6

The Nucleic Acid Sequence-Based Amplification (NASBA) process involves alternate steps of DNA synthesis from an RNA template and RNA synthesis from a DNA template, using avian myeloblastosis virus (AMV) reverse transcriptase and T7 RNA polymerase, respectively. The overall fidelity of the amplification process was determined by sequence analysis of cloned DNA products of NASBA reactions. An error frequency of less than 0.3% was observed in cloned DNA products from two different segments of the HIV-1 gag gene. Partial substitution of GTP with ITP in the NASBA reaction did not significantly change the fidelity of the process. An error rate of 2 x 10(-4) was calculated for the combined effects of both polymerases.
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PMID:Fidelity of nucleic acid amplification with avian myeloblastosis virus reverse transcriptase and T7 RNA polymerase. 753 77

Intrinsic termination of T7 RNA polymerase transcription occurs at different signals in vitro. One type of signal is similar to that mediating factor-independent termination of Escherichia coli RNA polymerase, whereas the other type does not involve RNA hairpin formation. By examining the termination behaviour of T7 RNA polymerase at the E.coli rrnB operon t1 terminator, at the T7-t(phi) terminator, at the human preproparathyroid hormone gene terminator on both single- and double-stranded templates, and in the presence of GTP or ITP during transcription, we show that the termination event can be mediated by either RNA or DNA structural features. Moreover, by using co-transcriptional probing with potassium permanganate, we present evidence for the presence of transcription-induced hyperreactive thymidines on the non-template strand in the DNA-mediated event, and a putative sequence motif is identified. We conclude that intrinsic termination of T7 RNA polymerase transcription in vitro can be mediated either by a hairpin in the nascent RNA or by a sequence motif including hyperreactive thymidines in the non-template DNA strand.
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PMID:Intrinsic termination of T7 RNA polymerase mediated by either RNA or DNA. 888 68


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