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)

To modulate transcription, regulatory factors communicate with basal transcription factors and/or RNA polymerases in a variety of ways. Previously, it has been reported that RNA polymerase II subunit 5 (RPB5) is one of the targets of hepatitis B virus X protein (HBx) and that both HBx and RPB5 specifically interact with general transcription factor IIB (TFIIB), implying that RPB5 is one of the communicating subunits of RNA polymerase II involved in transcriptional regulation. In this context, we screened for a host protein(s) that interacts with RPB5. By far-Western blot screening, we cloned a novel gene encoding a 508-amino-acid-residue RPB5-binding protein from a HepG2 cDNA library and designated it RPB5-mediating protein (RMP). Expression of RMP mRNA was detected ubiquitously in various tissues. Bacterially expressed recombinant RMP strongly bound RPB5 but neither HBx nor TATA-binding protein in vitro. Endogenous RMP was immunologically detected interacting with assembled RPB5 in RNA polymerase in mammalian cells. The central part of RMP is responsible for RPB5 binding, and the RMP-binding region covers both the TFIIB- and HBx-binding sites of RPB5. Overexpression of RMP, but not mutant RMP lacking the RPB5-binding region, inhibited HBx transactivation of reporters with different HBx-responsive cis elements in transiently transfected cells. The repression by RMP was counteracted by HBx in a dose-dependent manner. Furthermore, RMP has an inhibitory effect on transcriptional activation by VP16 in the absence of HBx. These results suggest that RMP negatively modulates RNA polymerase II function by binding to RPB5 and that HBx counteracts the negative role of RMP on transcription indirectly by interacting with RPB5.
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PMID:RMP, a novel RNA polymerase II subunit 5-interacting protein, counteracts transactivation by hepatitis B virus X protein. 981 40

The monoclonal antibody MPM-2 recognizes a subset of M phase phosphoproteins in a phosphorylation-dependent manner. It is believed that phosphorylation at MPM-2 antigenic sites could regulate mitotic events since most of the MPM-2 antigens identified to date have M phase functions. In addition, many of these proteins are substrates of the mitotic regulator Pin1, a peptidyl-prolyl isomerase which is present throughout the cell cycle and which is thought to alter its mitotic targets by changing their conformation. In interphase cells, most MPM-2 reactivity is confined to nuclear speckles. We report here that a hyperphosphorylated form of the RNA polymerase II largest subunit is the major MPM-2 interphase antigen. These findings were made possible by the availability of another monoclonal antibody, CC-3, that was previously used to identify a 255 kDa nuclear matrix protein associated with spliceosomal components as a hyperphosphorylated form of the RNA polymerase II largest subunit. MPM-2 recognizes a phosphoepitope of the large subunit that becomes hyperphosphorylated upon heat shock in contrast to the phosphoepitope defined by CC-3, whose reactivity is diminished by the heat treatment. Therefore, these two antibodies may discriminate between distinct functional forms of RNA polymerase II. We also show that RNA polymerase II large subunit interacts with Pin1 in HeLa cells. Pin1 may thus regulate transcriptional and post-transcriptional events by catalyzing phosphorylation-dependent conformational changes of the large RNA polymerase II subunit.
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PMID:A hyperphosphorylated form of RNA polymerase II is the major interphase antigen of the phosphoprotein antibody MPM-2 and interacts with the peptidyl-prolyl isomerase Pin1. 1039 5

CC3 is a metastasis suppressor that inhibits metastasis of the variant small cell lung carcinoma (v-SCLC) by predisposing cells to apoptosis. The same protein was also reported as a cellular cofactor, TIP30, which stimulates HIV-1 Tat-activated transcription by interacting with both Tat and RNA polymerase II. We report here that TIP30/CC3 is a novel serine/threonine kinase. It phosphorylates the heptapeptide repeats of the C-terminal domain (CTD) of the largest RNA polymerase II subunit in a Tat-dependent manner. Amino acid substitutions in the putative ATP binding motif that abolish the TIP30 kinase activity also inhibit the ability of TIP30 to enhance Tat-activated transcription or to sensitize NIH 3T3 and v-SCLC cells to apoptosis. Furthermore, ectopic expression of TIP30/CC3 in v-SCLC cells induces expression of a number of genes that include the apoptosis-related genes Bad and Siva, as well as metastasis suppressor NM23-H2. These data demonstrate a molecular mechanism for TIP30/CC3 function and suggest a novel pathway for regulating apoptosis.
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PMID:TIP30 has an intrinsic kinase activity required for up-regulation of a subset of apoptotic genes. 1069 37

Transcription factor IIB (TFIIB) is an essential component in the formation of the transcription initiation complex in eucaryal and archaeal transcription. TFIIB interacts with a promoter complex containing the TATA-binding protein (TBP) to facilitate interaction with RNA polymerase II (RNA pol II) and the associated transcription factor IIF (TFIIF). TFIIB contains a zinc-binding motif near the N-terminus that is directly involved in the interaction with RNA pol II/TFIIF and plays a crucial role in selecting the transcription initiation site. The solution structure of the N-terminal residues 2-59 of human TFIIB was determined by multidimensional NMR spectroscopy. The structure consists of a nearly tetrahedral Zn(Cys)3(His)1 site confined by type I and "rubredoxin" turns, three antiparallel beta-strands, and disordered loops. The structure is similar to the reported zinc-ribbon motifs in several transcription-related proteins from archaea and eucarya, including Pyrococcus furiosus transcription factor B (PfTFB), human and yeast transcription factor IIS (TFIIS), and Thermococcus celer RNA polymerase II subunit M (TcRPOM). The zinc-ribbon structure of TFIIB, in conjunction with the biochemical analyses, suggests that residues on the beta-sheet are involved in the interaction with RNA pol II/TFIIF, while the zinc-binding site may increase the stability of the beta-sheet.
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PMID:Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcription. 1104 20

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30 in vitro using purified recombinant proteins and in vivo in COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47-120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101-170) and the N-terminus (aa 1-100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding in in vitro and in vivo assays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.
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PMID:Direct interaction between the subunit RAP30 of transcription factor IIF (TFIIF) and RNA polymerase subunit 5, which contributes to the association between TFIIF and RNA polymerase II. 1127 33

We used whole genome expression analysis to investigate the changes in the mRNA profile in cells lacking the Saccharomyces cerevisiae RNA polymerase II subunit RPB4 (Delta RPB4). Our results indicated that an essentially complete shutdown of transcription occurs upon temperature shift of this conditionally lethal mutant; 98% of mRNA transcript levels decrease at least 2-fold, 96% at least 4-fold. This data was supported by in vivo experiments that revealed a rapid and greater than 5-fold decline in steady state poly(A) RNA levels after the temperature shift. Expression of several individual genes, measured by Northern analysis, was also consistent with the whole genome expression profile. Finally we demonstrated that the loss of RNA polymerase II activity causes secondary effects on RNA polymerase I, but not RNA polymerase III, transcription. The transcription phenotype of the Delta RPB4 mutant closely mirrors that of the temperature-sensitive rpb1-1 mutant frequently implemented as a tool to inactivate the RNA polymerase II in vivo. Therefore, the Delta RPB4 mutant can be used to easily design strains that enable the study of distinct post-transcriptional cellular processes in the absence of RNA polymerase II transcription.
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PMID:Deletion of the RNA polymerase subunit RPB4 acts as a global, not stress-specific, shut-off switch for RNA polymerase II transcription at high temperatures. 1157 1

The repetitive C-terminal domain (CTD) of the largest RNA polymerase II subunit plays a critical role in the regulation of gene expression. The activity of the CTD is dependent on its state of phosphorylation. A variety of CTD kinases act on RNA polymerase II at specific steps in the transcription cycle and preferentially phosphorylate distinct positions within the CTD consensus repeat. A single CTD phosphatase has been identified and characterized that in concert with CTD kinases establishes the level of CTD phosphorylation. The involvement of CTD phosphatase in controlling the progression of RNAP II around the transcription cycle, the mobilization of stored RNAP IIO, and the regulation of transcript elongation and RNA processing is discussed.
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PMID:CTD phosphatase: role in RNA polymerase II cycling and the regulation of transcript elongation. 1220 56

TFIIS, an elongation factor encoded by DST1 in Saccharomyces cerevisiae, stimulates transcript cleavage in arrested RNA polymerase II. Two components of the RNA polymerase II machinery, Med13 (Srb9) and Spt8, were isolated as two-hybrid partners of the conserved TFIIS N-terminal domain. They belong to the Cdk8 module of the Mediator and to a subform of the SAGA co-activator, respectively. Co-immunoprecipitation experiments showed that TFIIS can bind the Cdk8 module and SAGA in cell-free extracts. spt8Delta and dst1Delta mutants were sensitive to nucleotide-depleting drugs and epistatic to null mutants of the RNA polymerase II subunit Rpb9, suggesting that their elongation defects are mediated by Rpb9. rpb9Delta, spt8Delta and dst1Delta were lethal in cells lacking the Rpb4 subunit. The TFIIS N-terminal domain is also strictly required for viability in rpb4Delta, although it is not needed for binding to RNA polymerase II or for transcript cleavage. It is proposed that TFIIS and the Spt8-containing form of SAGA co-operate to rescue RNA polymerase II from unproductive elongation complexes, and that the Cdk8 module temporarily blocks transcription during transcript cleavage.
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PMID:Members of the SAGA and Mediator complexes are partners of the transcription elongation factor TFIIS. 1535 73

Precise control of gene expression is critical for embryo development in both animals and plants. We report that Arabidopsis thaliana GLUTAMINE-RICH PROTEIN23 (GRP23) is a pentatricopeptide repeat (PPR) protein that functions as a potential regulator of gene expression during early embryogenesis in Arabidopsis. Loss-of-function mutations of GRP23 caused the arrest of early embryo development. The vast majority of the mutant embryos arrested before the 16-cell dermatogen stage, and none of the grp23 embryos reached the heart stage. In addition, 19% of the mutant embryos displayed aberrant cell division patterns. GRP23 encodes a polypeptide with a Leu zipper domain, nine PPRs at the N terminus, and a Gln-rich C-terminal domain with an unusual WQQ repeat. GRP23 is a nuclear protein that physically interacts with RNA polymerase II subunit III in both yeast and plant cells. GRP23 is expressed in developing embryos up to the heart stage, as revealed by beta-glucuronidase reporter gene expression and RNA in situ hybridization. Together, our data suggest that GRP23, by interaction with RNA polymerase II, likely functions as a transcriptional regulator essential for early embryogenesis in Arabidopsis.
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PMID:Arabidopsis GLUTAMINE-RICH PROTEIN23 is essential for early embryogenesis and encodes a novel nuclear PPR motif protein that interacts with RNA polymerase II subunit III. 1648 21

Affinity tagging has been used in many global studies towards protein function. We describe a highly efficient system for in vivo biotinylation of transcription factors in the yeast Saccharomyces cerevisiae, which is based on the bacterial BirA biotin ligase. The strength of the biotin-streptavidin interaction was exploited to improve detection of in vivo protein-DNA complexes in chromatin immunoprecipitation (ChIP) experiments. In a test system using the biotin-tagged LexA DNA-binding protein, we found that stringent washing conditions resulted in a strong improvement of the signal-to-noise ratios. Yeast strains with chromosomally integrated versions of tagged transcription factor genes were generated using N- or C-terminal biotin-tagging cassettes. ChIP experiments with biotinylated Rbp3p, a RNA polymerase II subunit, showed that Rbp3p-binding could even be detected at weakly expressed genes. Other methods failed to detect RNA polymerase II binding at such genes. Our results show that biotinylation of yeast transcription factors improves the detection of in vivo protein-DNA complexes.
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PMID:The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation. 1650 Aug 88


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