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)

We have investigated whether the RNA polymerase III-driven transcription of eukaryotic tRNA genes can be regulated by the prokaryotic tetracycline operator-repressor system. The bacterial tet operator (tetO) was inserted at two different positions (-7 and -46) upstream of a tRNA(Glu) (amber) suppressor gene. Both constructs are transcribed in Saccharomyces cerevisiae and yield functional tRNAs as scored by suppression of an amber nonsense mutation in the met8-1 allele. Controlled expression of Tet repressor was achieved by fusing the bacterial tetR gene to the yeast gal1 promoter. This leads to expression of Tet repressor in yeast on galactose--but not on glucose--containing media. Regulation of the su-tRNA gene with the tetO fragment inserted at position -7 has been demonstrated. Under conditions which allow tetR expression, cells exhibit a met- phenotype. This methionine auxotrophy can be conditionally reverted to prototrophy by adding tetracycline. However, a su-tRNA gene with the tetO fragment inserted at position -46 cannot be repressed. Our results demonstrate clearly that the bacterial repressor protein binds to its operator in the yeast genome. Formation of this complex in the vicinity of the pol III transcription initiation site reduces the level of su-tRNA at least 50-fold as concluded from quantitative primer extension analyses. This indicates for the first time that class III gene expression can be regulated by a DNA binding protein with its target site in the 5'-flanking region and that a prokaryotic repressor can confer regulation of a suitably engineered tRNA gene.
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PMID:RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system. 156 52

Using temperature- and proteolytically sensitive derivatives to inactivate the function of the yeast TATA-binding protein (TBP) in vivo, we investigated the requirement of TBP for transcription by the three nuclear RNA polymerases in yeast cells. TBP is required for RNA polymerase II (pol II) transcription from promoters containing conventional TATA elements as well as functionally distinct promoters that lack TATA-like sequences. TBP is also required for transcription of the U6 snRNA and two different tRNA genes mediated by RNA pol III as well as transcription of ribosomal RNA mediated by RNA pol I. For all promoters tested, transcription decreases rapidly and specifically upon inactivation of TBP, strongly suggesting that TBP is directly involved in the transcription process. These observations suggest that TBP is required for transcription of all nuclearly encoded genes in yeast, although distinct molecular mechanisms are probably involved for the three RNA polymerase transcription machineries.
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PMID:The TATA-binding protein is required for transcription by all three nuclear RNA polymerases in yeast cells. 158 47

UBF is a DNA binding protein which interacts with both the promoter and the enhancer of various vertebrate ribosomal RNA genes and functions as a transcription initiation factor for RNA polymerase I (pol I). We have purified murine UBF to apparent molecular homogeneity and demonstrate that its transactivating potential, but not its DNA binding activity, is modulated in response to cell growth. In vivo labelling experiments demonstrate that UBF is a phosphoprotein and that the phosphorylation state is different in growing and quiescent cells. We show that UBF is phosphorylated in vitro by a cellular protein kinase which by several criteria closely resembles casein kinase II (CKII). A major modification involves serine phosphoesterifications in the carboxy terminal hyperacidic tail of UBF. Deletions of this C-terminal domain severely decreases the UBF directed activation of transcription. The data suggest that phosphorylation of UBF by CKII may play an important role in growth dependent control of rRNA synthesis.
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PMID:The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation. 160 Sep 46

PCF1-1 is a dominant suppressor of a tRNA gene A block promoter mutation (A19) in Saccharomyces cerevisiae. Transcriptional activation by PCF1-1 was examined in vitro using whole-cell extracts and purified factors derived from mutant and wild-type strains. These experiments show that PCF1 is a general activator of RNA polymerase III (pol III) gene transcription. The transcription of all pol III genes analyzed to date, including type I and numerous type II genes, is increased 3-7 fold in mutant cell extracts. Single round transcription assays indicate that the PCF1-1 mutation increases the number of functional preinitiation complexes and suggest that this is achieved by increasing the intrinsic activity of the encoded product rather than its amount. Point mutations throughout the A block of the sup3-e gene and numerous B block mutations fail to abolish transcriptional activation suggesting that interactions between TFIIIC and the internal promoter are unaffected by PCF1-1. Moreover, TFIIIC purified from the mutant strain is incapable of conferring PCF1-1 transcriptional activity to a reaction in which the remaining components are wild-type. In contrast, the activity of the TFIIIB fraction is increased in PCF1-1 extracts and can reconstitute mutant levels of transcription when added to wild-type TFIIIC and polymerase. We conclude that PCF1 is a component or regulator of TFIIIB.
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PMID:The PCF1-1 mutation increases the activity of the transcription factor (TF) IIIB fraction from Saccharomyces cerevisiae. 164 38

Termination of transcription not only allows polymerases that have completed RNA synthesis to recycle, but it also has important functions in transcriptional regulation and in preventing promoter interference. The molecular basis for termination by RNA polymerase II (pol II) is unclear, however. We have identified a termination site in the promoter region of the c-myc gene, whose function correlates with DNA binding by a nuclear factor. When the c-myc gene was transcribed in injected Xenopus oocytes or a HeLa nuclear extract, a fraction of RNA initiated at the first promoter, P1, terminated at two positions, T1A and T1B, which flank the TATA box of the second promoter, P2. T1B is a T-rich sequence that resembles previously identified attenuation sites, but T1A appears to represent a different class of termination site. T1A is situated approximately 10 bases upstream of an element that overlaps the P2 TATA box. Mutagenesis of this element affected both the efficiency and the position at which termination occurred. A 28-base sequence including this element caused a low level of termination when inserted into the alpha-globin gene in either orientation. This sequence bound a factor called TBF I (terminator-binding factor), whose binding specificity correlated with T1A terminator function. We suggest that TBF I may function as a pol II termination factor.
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PMID:A protein-binding site in the c-myc promoter functions as a terminator of RNA polymerase II transcription. 164 97

We have combined immunogold labeling with the Miller spreading technique in order to localize proteins at the electron microscope (EM) level in whole mount nuclei from mouse and human fibroblasts. Anti-histone H1 antibody labels nuclei uniformly, indicating that the nuclear interior is accessible to both antibodies and gold conjugates. Anti-topoisomerase I antibody labels nucleoli intensely, in agreement with previous immunofluorescent and biochemical data. Two different antibodies against the large subunit of RNA polymerase II (pol II) show preferential labeling of the nuclear periphery, as do antibodies against lamin, a known peripheral nuclear protein. Treatment of cells with alpha-amanitin results in loss of virtually all RNA polymerase II staining, supporting the specificity of labeling. Finally, when nuclei are incubated in the presence of biotin-UTP (bio-UTP) under run-off transcription conditions, incorporation is preferentially located at the nuclear periphery. These results support the conclusions that transcriptionally active pol II molecules are non-uniformly distributed in fibroblast nuclei, and that their differential distribution mirrors that of total pol II.
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PMID:Preferential distribution of active RNA polymerase II molecules in the nuclear periphery. 166 44

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.
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PMID:Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. 168 98

Twelve to twenty percent of newly synthesized poly(A) + RNA is transcribed by RNA polymerase III in Ehrlich ascites carcinoma and P3O1 plasmocytoma mouse tumors. Most of this RNA designated as pol IIIpoly(A) + RNA has a size of 160 to 800 nucleotides with a maximum of distribution of ca. 300 nucleotides. Pol IIIpoly(A) + RNA fraction consists of two major classes of molecules corresponding to previously described B1 RNA and B2 RNA with the ratio of 1:4 to 2:3. All B2 RNAs present in poly(A) + fraction contain a long poly(A) segments at the 3' ends. Thus, RNA polymerase III transcripts can be polyadenylated. Several transcripts that hybridize with B2 probe were also observed in poly(A)- RNA. The major components consist of 180, 160, 120 and 95 nucleotides. The 180-nucleotide B2 RNA seems to be a primary transcript from B2 repeat. We suggest that other B2 RNAs are transcribed from truncated copies of B2 element.
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PMID:The most abundant nascent poly(A) + RNAs are transcribed by RNA polymerase III in murine tumor cells. 169 65

Previous studies revealed that antisense oligodeoxynucleotides to specific regions of the human immunodeficiency virus-1 (HIV-1) are potent inhibitors of replication of HIV-1 in vitro (Zamecnik, P. C., Goodchild, J., Taguchi, Y., and Sarin, P. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4143-4146). We now report that antisense RNA, synthesized in vitro using T7 and SP6 RNA polymerase, displayed an anti-HIV-1 effect in the HTLV-IIIB/H9 system in vitro. Treatment of HIV-1-infected H9 cells with viral env region antisense RNA encapsulated in liposomes targeted by antibodies specific for the T cell receptor molecule CD3 almost completely inhibited HIV-1 production. The viral env segment covered a part of exon II of HIV-1 tat gene. No anti-HIV activity could be detected with similarly targeted liposome-encapsulated sense env RNA or with pol RNA synthesized in either the sense or antisense orientations, or with env region antisense RNA free in solution, or encapsulated in liposomes in the absence of the targeting antibody. A semiquantitative evaluation revealed that 4000-7000 RNA molecules became cell-bound in targeted liposomes; the half-life of the intracellularly present hybridizable antisense env RNA was approximately 12 h. Western blots showed that antisense env RNA suppressed tat gene expression by approximately 90% and gp160 production by 100%. These data were confirmed by immunoprecipitation studies. Northern blots (using an env probe) demonstrated the existence of all major HIV RNA species (9.3-, 4.3-, and 2.0-kb mRNA) in HIV-infected cells treated with antisense env RNA although at a reduced level. We conclude that the antisense env RNA inhibited viral protein production at the translational level.
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PMID:Inhibition of expression of human immunodeficiency virus-1 in vitro by antibody-targeted liposomes containing antisense RNA to the env region. 169 56

The structural and enzymatic components of retroviral cores are formed by proteolytic cleavage of precursor polypeptides, mediated by the viral protease (PR). We constructed an active-site mutation, D37I, in the PR of avian leukosis virus. The D37I mutation was introduced into an infectious DNA clone, and quail cell lines expressing the mutant virus were established. These cell lines produce normal amounts of virus particles, the major internal protein components of which are the uncleaved gag and gag-pol precursors. As in other retroviral systems, the protease-defective virions are noninfectious and retain the "immature" type A morphology as determined by thin-section transmission electron microscopy. The virion cores are stable at nonionic detergent concentrations that completely disrupt wild-type cores. Digestion of mutant virions with exogenous PR in the presence of detergent leads to complete and correct cleavage of the gag precursor but incomplete cleavage of the gag-pol precursor. The protease-defective virions encapsidate normal amounts of genomic RNA and tRNA(Trp) that is properly annealed to the primer-binding site, but some of the genomic RNA remains monomeric. Results from UV cross-linking experiments show that the gag polyprotein of mutant virions interacts with viral RNA and that this interaction occurs through the nucleocapsid (NC) domain. However, within mutant virions the interaction of the NC domain with RNA differs from that of mature NC with RNA in wild-type virions. Reverse transcriptase (RT) activity associated with mutant virions is diminished but still detectable. Digestion of the virions with PR leads to a fivefold increase in activity, but this PR-mediated activation of RT is incomplete. Since in vitro cleavage of the gag-pol precursor is also incomplete, we hypothesize that amino acid sequences N terminal to the reverse transcriptase domain inhibit RT activity.
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PMID:Properties of avian retrovirus particles defective in viral protease. 169 12


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