Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA polymerase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

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

At least 55 records · Page 3Linked to original sources
PubMed1995

Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes.

The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP-TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific as gene-specific factors for PSE-and B-box-containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the from used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.

Base Sequence↗
PubMed2003

Behavior of T7 RNA polymerase and mammalian RNA polymerase II at site-specific cisplatin adducts in the template DNA.

Transcription-coupled DNA repair is dedicated to the removal of DNA lesions from transcribed strands of expressed genes. RNA polymerase arrest at a lesion has been proposed as a sensitive signal for recruitment of repair enzymes to the lesion site. To understand how initiation of transcription-coupled repair may occur, we have characterized the properties of the transcription complex when it encounters a lesion in its path. Here we have compared the effect of cisplatin-induced intrastrand cross-links on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We found that a single cisplatin 1,2-d(GG) intrastrand cross-link or a single cisplatin 1,3-d(GTG) intrastrand cross-link is a strong block to both polymerases. Furthermore, the efficiency of the block at a cisplatin 1,2-d(GG) intrastrand cross-link was similar in several different nucleotide sequence contexts. Interestingly, some blockage was also observed when the single cisplatin 1,3-d(GTG) intrastrand cross-link was located in the non-transcribed strand. Transcription complexes arrested at the cisplatin adducts were substrates for the transcript cleavage reaction mediated by the elongation factor TFIIS, indicating that the RNA polymerase II complexes arrested at these lesions are not released from template DNA. Addition of TFIIS yielded a population of transcripts up to 30 nucleotides shorter than those arrested at the lesion. In the presence of nucleoside triphosphates, these shortened transcripts could be re-elongated up to the site of the lesion, indicating that the arrested complexes are stable and competent to resume elongation. These results show that cisplatin-induced lesions in the transcribed DNA strand constitute a strong physical barrier to RNA polymerase progression, and they support current models of transcription arrest and initiation of transcription-coupled repair.

3' Untranslated Regions↗
PubMed1981

A direct effect of guanosine tetraphosphate on pausing of Escherichia coli RNA polymerase during RNA chain elongation.

The effect of the regulatory nucleotide ppGpp on transcription by Escherichia coli RNA polymerase in vitro has been studied using bacteriophage T7 and T3 DNAs as templates. We have previously described the development of transcriptional test systems using these templates that can sensitively detect changes in promotor or terminator recognition of an RNA polymerase (Wiggs, J.L., Bush, J.W., and Chamberlin, M.J. (1979) Cell 16, 97-109) or changes in the rate of any of the major steps of the transcription cycle (Chamberlin, M.J., Nierman, W.C., Wiggs, J.L., and Neff, N. (1979) J. Biol. Chem. 254, 10061-10069). Using these procedures we fail to detect any substantial alteration by ppGpp of the normal interaction of E. coli RNA polymerase with the several T7 major and minor promoter sites or of the rate of productive RNA chain initiation at either T7 promoter A1 (ATP start) or A2 (GTP start). However, at physiologically relevant concentrations (KI approximately 50 microM), ppGpp significantly lowers the overall elongation rate of T7 RNA chains, leading to a substantial reduction in the overall rate of RNA synthesis. Inhibition of transcriptional elongation does not appear to be competitive with the ribonucleoside triphosphate substrates. Furthermore, ppGpp does not inhibit chain elongation during transcription of the synthetic polynucleotide templates poly[d(A-T)] or poly[d(A-G):d(C-T)]. We conclude that ppGpp interacts directly with some site on RNA polymerase other than one of the sites used for substrate binding. Furthermore, the inhibition must depend on specific DNA sequences present in T7 DNA, but not in poly[d(A-T)] or poly[d(A-G):d(C-T)]. Analysis of T7 transcripts formed in the presence and absence of ppGpp by gel electrophoresis reveals that in the latter instance there is enhanced pausing of the transcriptional elongation complex at specific sites on the template. It is likely that the enzyme also pauses at such sequences in the absence of ppGpp, but for a far briefer time (Kassavetis, G.A., and Chamberlin, M.J. (1981) J. Biol. Chem. 256, 2777-2786). Thus, ppGpp appears to slow transcriptional elongation by binding to RNA polymerase and altering its structure in a manner that impedes passage of the enzyme through certain DNA sequences. The presence of similar transcriptional barriers in rRNA operons activated by the presence of ppGpp could lead selectively to large reductions in the rate of rRNA synthesis in vivo.

DNA, Viral↗
PubMed1981

Localization of the Tn5 transposase promoter using the cycling reaction of RNA polymerase.

RNA polymerase is known to undergo a cycling process during initiation of transcription in vitro in which large amounts of small oligonucleotides are released. We have used this cycling reaction to determine the 5' end of the RNA synthesized from the outer ends of the Tn5 inverted repeats. By analyzing the size of the radiolabeled oligonucleotides synthesized using different labeled nucleoside triphosphates and in reactions deficient for a particular nucleoside triphosphate, the partial 5' sequence was obtained. This sequence was correlated with the DNA sequence of the region and an unambiguous origin for the mRNA was determined. The start site for the RNA, which is located at 97 base pairs from the outer ends of the inverted repeats, was confirmed by digesting gamma-32 P-ATP labeled full length (run off) transcripts with ribonuclease T1. The resulting gamma-labeled T1 generated oligonucleotide corresponded to the predicted size determined using the cycling reaction. With the knowledge of the RNA start site, the probable translation start sites for the protein(s) known to be required for Tn5 transposition can be predicted. Possible implications of the DNA sequence with respect to the regulation of the transposase are also discussed.

Base Sequence↗
PubMed1996

Base-specific recognition of the nontemplate strand of promoter DNA by E. coli RNA polymerase.

RNA polymerase recognizes its promoters through base-specific interaction between defined segments of DNA and the sigma subunit of the enzyme. This interaction leads to separation of base pairs and exposure of the template strand for RNA synthesis. We show that base-specific recognition by the sigma 70 holoenzyme in this process involves primarily nontemplate strand bases in the -10 promoter region. We suggest that melting involves the persistence of these contacts as the bound duplex (closed) form is converted to the single-stranded (open) form of the enzyme-promoter complex.

Base Sequence↗
PubMed1998

Force and velocity measured for single molecules of RNA polymerase.

RNA polymerase (RNAP) moves along DNA while carrying out transcription, acting as a molecular motor. Transcriptional velocities for single molecules of Escherichia coli RNAP were measured as progressively larger forces were applied by a feedback-controlled optical trap. The shapes of RNAP force-velocity curves are distinct from those of the motor enzymes myosin or kinesin, and indicate that biochemical steps limiting transcription rates at low loads do not generate movement. Modeling the data suggests that high loads may halt RNAP by promoting a structural change which moves all or part of the enzyme backwards through a comparatively large distance, corresponding to 5 to 10 base pairs. This contrasts with previous models that assumed force acts directly upon a single-base translocation step.

DNA, Bacterial↗
PubMed1998

Force generation in RNA polymerase.

RNA polymerase (RNAP) is a processive molecular motor capable of generating forces of 25-30 pN, far in excess of any other known ATPase. This force derives from the hydrolysis free energy of nucleotides as they are incorporated into the growing RNA chain. The velocity of procession is limited by the rate of pyrophosphate release. Here we demonstrate how nucleotide triphosphate binding free energy can rectify the diffusion of RNAP, and show that this is sufficient to account for the quantitative features of the measured load-velocity curve. Predictions are made for the effect of changing pyrophosphate and nucleotide concentrations and for the statistical behavior of the system.

Adenosine Triphosphatases↗
PubMed2004

Effect of 8-oxoguanine on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II.

8-Oxoguanine (8-oxoG) is a major oxidative lesion produced in DNA by normal cellular metabolism or after exposure to exogenous sources such as ionizing radiation. Persistence of this lesion in DNA causes G to T transversions, with deleterious consequences for the cell. As a result, several repair processes have evolved to remove this lesion from the genome. It has been reported that 8-oxoG is subject to transcription-coupled repair (TCR), a process dedicated to removal of lesions from transcribed strands of expressed genes. A current model assumes that RNA polymerase arrest at the site of the lesion is required for initiation of TCR. As a first step to understand how TCR of 8-oxoG occurs, we have studied the effect of 8-oxoG on transcription elongation by T7 RNA polymerase (T7 RNAP) and rat liver RNA polymerase II (RNAPII). We have utilized an in vitro transcription system with purified RNA polymerase and initiation factors, and substrates containing a single 8-oxoG in the transcribed or in the non-transcribed strand downstream of the T7 promoter or the Adenovirus major late promoter. We found that 8-oxoG only slightly inhibited T7 RNAP transcription, with a readthrough frequency of up to 95%. Similarly, this lesion only transiently blocked transcription by RNAPII. However, changes in nucleotide concentration affected the extent of RNAPII blockage at the 8-oxoG. When this lesion was positioned in the non-transcribed strand, complete lesion bypass was observed with either polymerase. Binding of the Saccharomyces cerevisiae MSH2-MSH6 complex to 8-oxoG containing substrates did not increase the frequency of RNAPII arrest at the site of the lesion, suggesting that this complex was displaced by the elongating polymerase. These results are discussed in the context of possible models for TCR.

Animals↗
PubMed1999

Mutants in ABC10beta, a conserved subunit shared by all three yeast RNA polymerases, specifically affect RNA polymerase I assembly.

ABC10beta, a small polypeptide common to the three yeast RNA polymerases, has close homology to the N subunit of the archaeal enzyme and is remotely related to the smallest subunit of vaccinial RNA polymerase. The eucaryotic, archaeal, and viral polypeptides share an invariant motif CX2C. CC that is strictly essential for yeast growth, as shown by site-directed mutagenesis, whereas the rest of the ABC10beta sequence is fairly tolerant to amino acid replacements. ABC10beta has Zn2+ binding properties in vitro, and the CX2C. CC motif may therefore define an atypical metal-chelating site. Hybrid subunits that derive most of their amino acids from the archaeal subunit are functional in yeast, indicating that the archaeal and eucaryotic polypeptides have a largely equivalent role in the organization of their respective transcription complexes. However, all eucaryotic forms of ABC10beta harbor a HVDLIEK motif that, when mutated or replaced by its archaeal counterpart, leads to a polymerase I-specific lethal defect in vivo. This is accompanied by a specific lack in the largest subunit of RNA polymerase I (A190) in cell-free extracts, showing that the mutant enzyme is not properly assembled in vivo.

Amino Acid Sequence↗
PubMed1997

Histones H3 and H4 are components of upstream activation factor required for the high-level transcription of yeast rDNA by RNA polymerase I.

RNA polymerase I (Pol I) transcription in the yeast Saccharomyces cerevisiae is greatly stimulated in vivo and in vitro by the multiprotein complex, upstream activation factor (UAF). UAF binds tightly to the upstream element of the rDNA promoter, such that once bound (in vitro), UAF does not readily exchange onto a competing template. Of the polypeptides previously identified in purified UAF, three are encoded by genes required for Pol I transcription in vivo: RRN5, RRN9, and RRN10. Two others, p30 and p18, have remained uncharacterized. We report here that the N-terminal amino acid sequence, its mobility in gel electrophoresis, and the immunoreactivity of p18 shows that it is histone H3. In addition, histone H4 was found in UAF, and myc-tagged histone H4 could be used to affinity-purify UAF. Histones H2A and H2B were not detectable in UAF. These results suggest that histones H3 and H4 probably account for the strong binding of UAF to DNA and may offer a means by which general nuclear regulatory signals could be transmitted to Pol I.

Amino Acid Sequence↗
PubMed1985

Intranuclear compartmentalization of DNA-dependent RNA polymerases: association of RNA polymerase I with nucleolar organizing chromosomes.

The compartmentalization of chromatin-bound RNA polymerases was examined with HeLa chromosomes fractionated according to their size in sucrose/glycerol gradients. There was a good correlation between the enrichment of nucleolus-organizing chromosomes, i.e., D and G group chromosomes, and the level of chromosome-associated RNA polymerase form I activity. A profile of DEAE-Sephadex chromatography of enzymes solubilized from chromosome fractions also supported the view that form I was associated with D and G group chromosomes. The amount of form I associated with unfractionated chromosomes was nearly identical to that of nuclei, indicating that this enzyme is probably quantitatively conserved even when chromatin undergoes extensive condensation. Since the chromatin-bound form I enzyme can be reactivated with heparin, it seems that this enzyme is in the initiated state, probably being bound to rDNA throughout the mitotic cycle. Thus, the absence of rRNA synthesis in mitosis is due to neither unavailability of enzyme to rDNA nor to the release of some factors necessary for transcriptional processes. Form II enzyme was associated uniformly with all chromosome fractions. Taken together, the present findings suggest that the intranuclear compartmentalization of RNA polymerases persists not only in interphase stages, but also in mitosis, during which most other nuclear proteins are released into the cytoplasm.

Cell Fractionation↗
PubMed1995

Both RNA polymerase III and RNA polymerase II accurately initiate transcription from a human U6 promoter in vitro.

The promoter of vertebrate U6 small nuclear RNA genes consists of a TATA box and a snRNA proximal sequence element (PSE), and the combination of these two elements directs RNA polymerase III transcription. We detected RNA polymerase II transcription as well as pol III transcription from the human U6 promoter in a HeLa nuclear extract. The pol II-specific transcription was independent of the PSE and dependent on the presence of the TATA box. Both pol III- and pol II-specific transcription were stimulated by addition of recombinant TATA-binding protein (TBP). We conclude that both pol III and pol II preinitiation complexes can assemble on the U6 promoter in vitro and could compete during the bona fide process in the cell.

Animals↗
PubMed1981

Identification of transcription initiation sites for bacterial RNA polymerase and eukaryotic RNA polymerase B on the 5' end of the mouse beta-Globin gene.

Using a recombinant phage containing the mouse beta-Globin gene with lambda gtWES bacteriophage DNA, transcription initiation sites for Escherichia coli RNA polymerase and calf thymus RNA polymerase B were mapped at the 5' and 3' ends of the mouse beta-Globin gene. The bacterial enzyme was capable of initiating RNA synthesis at the 3' end site located at about 700 residues from the 3' end of the beta-Globin restriction enzyme map. Initiation at this site was more efficient than initiation at the known early lambda promotors (PL, PR). Calf thymus RNA polymerase B initiated transcription at the same sites as the bacterial enzyme but in this case maximum efficiency was at the 5' end site as compared to the 3' end site. Initiation of transcription occurs in the region of the d(T-A-T-A-A) sequence. Initiation efficiency at the 5' end site, as probed by the maximum rate of transcription, was shown to depend partly upon the presence of the adjacent sequences upstream and downstream of the 5' initiation site.

Base Sequence↗
PubMed1994

Small nuclear RNA genes transcribed by either RNA polymerase II or RNA polymerase III in monocot plants share three promoter elements and use a strategy to regulate gene expression different from that used by their dicot plant counterparts.

RNA polymerase (Pol) II- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes of dicotyledonous plants contain two essential upstream promoter elements, the USE and TATA. The USE is a highly conserved plant snRNA gene-specific element, and its distance from the -30 TATA box, corresponding to approximately three and four helical DNA turns in Pol III and Pol II genes, respectively, is crucial for determining RNA Pol specificity of transcription. Sequences upstream of the USE play no role in snRNA gene transcription in dicot plants. Here we show that for expression of snRNA genes in maize, a monocotyledonous plant, the USE and TATA elements are essential, but not sufficient, for transcription. Efficient expression of both Pol II- and Pol III-specific snRNA genes in transfected maize protoplasts requires an additional element(s) positioned upstream of the USE. This element, named MSP (for monocot-specific promoter; consensus, RGCCCR), is present in one to three copies in monocot snRNA genes and is interchangeable between Pol II- and Pol III-specific genes. The efficiency of snRNA gene expression in maize protoplast is determined primarily by the strength of the MSP element(s); this contrasts with the situation in protoplasts of a dicot plant, Nicotiana plumbaginifolia, where promoter strength is a function of the quality of the USE element. Interestingly, the organization of monocot Pol III-specific snRNA gene promoters closely resembles those of equivalent vertebrate promoters. The data are discussed in the context of the coevolution of Pol II- and Pol III-specific snRNA gene promoters within many eukaryotic organisms.

Arabidopsis↗
PubMed2000

The alpha subunit of E. coli RNA polymerase activates RNA binding by NusA.

The Escherichia coli NusA protein modulates pausing, termination, and antitermination by associating with the transcribing RNA polymerase core enzyme. NusA can be covalently cross-linked to nascent RNA within a transcription complex, but does not bind RNA on its own. We have found that deletion of the 79 carboxy-terminal amino acids of the 495-amino-acid NusA protein allows NusA to bind RNA in gel mobility shift assays. The carboxy-terminal domain (CTD) of the alpha subunit of RNA polymerase, as well as the bacteriophage lambda N gene antiterminator protein, bind to carboxy-terminal regions of NusA and enable full-length NusA to bind RNA. Binding of NusA to RNA in the presence of alpha or N involves an amino-terminal S1 homology region that is otherwise inactive in full-length NusA. The interaction of the alpha-CTD with full-length NusA stimulates termination. N may prevent termination by inducing NusA to interact with N utilization (nut) site RNA rather than RNA near the 3' end of the nascent transcript. Sequence analysis showed that the alpha-CTD contains a modified helix-hairpin-helix motif (HhH), which is also conserved in the carboxy-terminal regions of some eubacterial NusA proteins. These HhH motifs may mediate protein-protein interactions in NusA and the alpha-CTD.

Amino Acid Motifs↗
PubMed1984

The mechanism of pyrophosphorolysis of RNA by RNA polymerase. Endowment of RNA polymerase with artificial exonuclease activity.

DNA-directed RNA polymerase from Escherichia coli can break down RNA by catalysing the reverse of the reaction: NTP + (RNA)n = (RNA)n+1 + PPi where n indicates the number of nucleotide residues in the RNA molecule, to yield nucleoside triphosphates. This reaction requires the ternary complex of the polymerase with template DNA and the RNA that it has synthesized. It is now shown that methylenebis(arsonic acid) [CH2(AsO3H2)2], arsonomethylphosphonic acid (H2O3As-CH2-PO3H2) and arsonoacetic acid (H2O3As-CH2-CO2H) can replace pyrophosphate in this reaction. When they do so, the low-Mr products of the reaction prove to be nucleoside 5'-phosphates, so that the arsenical compounds endow the polymerase with an artificial exonuclease activity, an effect previously found by Rozovskaya, Chenchik, Tarusova, Bibilashvili & Khomutov [(1981) Mol. Biol. (Moscow) 15, 636-652] for phosphonoacetic acid (H2O3P-CH2-CO2H). This is explained by instability of the analogues of nucleoside triphosphates believed to be the initial products. Specificity of recognition of pyrophosphate is discussed in terms of the sites, beta and gamma, for the -PO3H2 groups of pyrophosphate that will yield P-beta and P-gamma of the nascent nucleoside triphosphate. Site gamma can accept -AsO3H2 in place of -PO3H2, but less well; site beta can accept both, and also -CO2H. We suggest that partial transfer of an Mg2+ ion from the attacking pyrophosphate to the phosphate of the internucleotide bond of the RNA may increase the nucleophilic reactivity of the pyrophosphate and the electrophilicity of the diester, so that the reaction is assisted.

Binding Sites↗
PubMed1999

Mutations in conserved domain II of the large (L) subunit of the Sendai virus RNA polymerase abolish RNA synthesis.

The large (L) protein of Sendai virus complexes with the phosphoprotein (P) to form the active RNA-dependent RNA polymerase. The L protein is believed to be responsible for all of the catalytic activities of the polymerase associated with transcription and replication. Sequence alignment of the L proteins of negative-strand RNA viruses has revealed six conserved domains (I-VI) thought to be responsible for the enzymatic activities. Charged-to-alanine mutagenesis was carried out in a highly charged, conserved region (amino acids 533-569) within domain II to test the hypothesis of Müller et al. [J. Gen. Virol. 75, 1345-1352 (1994)] that this region may contribute to the template binding domain of the viral RNA polymerase. The mutant proteins were tested for expression and stability, the ability to synthesize viral RNA in vitro and in vivo, and protein-protein interactions. Five of the seven mutants were completely defective in all viral RNA synthesis, whereas two mutants showed significant levels of both mRNA and leader RNA synthesis. One of the transcriptionally active mutants also gave genome replication in vitro although not in vivo. The other mutant was defective in all the replication assays and thus the mutation uncoupled transcription and replication. Because the completely inactive L mutants can bind to the P protein to form the polymerase complex and the polymerases bind to the viral nucleocapsid template, these amino acids are essential for the activity of the L protein.

Amino Acid Sequence↗
PubMed2001

RNA polymerase III and RNA polymerase II promoter complexes are heterochromatin barriers in Saccharomyces cerevisiae.

The chromosomes of eukaryotes are organized into structurally and functionally discrete domains. Several DNA elements have been identified that act to separate these chromatin domains. We report a detailed characterization of one of these elements, identifying it as a unique tRNA gene possessing the ability to block the spread of silent chromatin in Saccharomyces cerevisiae efficiently. Transcriptional potential of the tRNA gene is critical for barrier activity, as mutations in the tRNA promoter elements, or in extragenic loci that inhibit RNA polymerase III complex assembly, reduce barrier activity. Also, we have reconstituted the Drosophila gypsy element as a heterochromatin barrier in yeast, and have identified other yeast sequences, including the CHA1 upstream activating sequence, that function as barrier elements. Extragenic mutations in the acetyltransferase genes SAS2 and GCN5 also reduce tRNA barrier activity, and tethering of a GAL4/SAS2 fusion creates a robust barrier. We propose that silencing mediated by the Sir proteins competes with barrier element-associated chromatin remodeling activity.

Acetyltransferases↗