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)

Identification of the RNA polymerase functional regions involved in interactions with promoter is a basis for understanding the mechanism of transcription initiation. We have used formaldehyde cross-linking to identify a region of Escherichia coli RNA polymerase beta' subunit contacting lacUV5 promoter in open complex. Treatment of open complex with formaldehyde results in cross-linking of beta' and sigma(70) subunits at positions -5 and -3 on the nontemplate strand of the promoter DNA. These cross-links reflect specific interactions between RNA polymerase and promoter established in open complex. The positions of formaldehyde cross-links in the beta' subunit were mapped to the N-terminal segment (Cys(198)-Met(237)), which is contiguous to the evolutionary conserved region B. The proximity of the beta' and sigma cross-links suggest that the N-terminal region of the beta' subunit, interacting with single-stranded promoter DNA, can cooperate with the sigma subunit in the process of open complex formation.
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PMID:Identification of RNA polymerase beta' subunit segment contacting the melted region of the lacUV5 promoter. 1065 63

Transcription of TATA box-containing genes by RNA polymerase II is mediated by TBP-containing and TBP-free multisubunit complexes consisting of common and unique components. We have identified a highly stable TBP-TFIIA-containing complex, TAC, which is detectable in embryonal carcinoma (EC) cells but not in differentiated cells. TAC contains the TFIIAgamma subunit and the unprocessed form of TFIIAalphabeta, although the processed TFIIAalpha and TFIIAbeta subunits are present in EC cells. TAC mediates transcriptional activation by RNA polymerase II in vivo, even though it does not contain classical TAFs. Formaldehyde cross-linking revealed that in EC but not in differentiated cells, association of TBP with chromatin is strongly enhanced when complexed with TFIIA in vivo. Remarkably, the TFIIAalphabeta precursor is preferentially, if not exclusively, associated with chromatin as compared to the processed subunits present in "free" TFIIA in EC cells.
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PMID:TAC, a TBP-sans-TAFs complex containing the unprocessed TFIIAalphabeta precursor and the TFIIAgamma subunit. 1103 Mar 33

Formaldehyde cross-linking was used in a kinetic analysis of RNA polymerase-lacUV5 promoter interactions in open complexes (RP(o)). RP(o) quenched from 37 degrees C to 14 degrees C isomerised to a closed, competitor resistant, complex (RP(LT)). We observed that contacts of the beta' and sigma subunits with the positions -3, -5 of the non-template DNA strand disappeared very quickly during the first 30 seconds after the temperature downshift. The re-annealing of the DNA downstream of the transcription start site takes place in the same time scale. However re-annealing of the upstream part of the transcription bubble was slower and completed within five minutes. The results support a two-step model of promoter melting and suggest that conformational changes in the RNA polymerase occur concurrently with the melting around the transcription start site.
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PMID:Differential melting of the transcription start site associated with changes in RNA polymerase-promoter contacts in initiating transcription complexes. 1124

We established an in vitro system representing BL-type EBV infection, which is characterized by expression of EBNA1, EBER, BARF0, and LMP2A, and absence of EBNA2 and LMP1 expression (Shimizu et al. 1994; Komano et al. 1998). Comparison of EBV-positive and -negative Akata cell clones revealed that EBV contributes to the malignant phenotype and resistance to apoptosis. This is clear evidence that EBV is not a passenger and plays a role in BL. Moreover, we found that EBERs are responsible for these phenotypes (Komano et al. 1999). In the transfection study, EBER-expressing Akata cell clones restored the malignant phenotype, resistance to apoptosis and upregulated expression of bcl-2 protein to a level comparable to the restoration rate of EBER expression compared with EBV-reinfected cell clones. Many RNAs are known to have catalytic functions; however, there has been no report describing an oncogenic RNA. This is the first paper that provides evidence that RNA polymerase III-transcribed virus-encoded small RNAs affect the malignant phenotype and resistance to apoptosis. Like Akata cells (Takada et al. 1991), all the BL cells possess a chromosomal translocation involving the c-myc locus, which results in constitutive activation of the c-myc gene (Klein 1981). In mammalian cells, deregulated expression of c-myc has been shown to contribute not only to tumorigenesis (Land et al. 1983) but also to induce apoptosis (Askew et al. 1991; Evan et al. 1992; Milner et al. 1993). Therefore, BL cells are predisposed to c-myc-induced apoptosis. Our data imply that EBV infection would upregulate expression of bcl-2 protein to protect cells from c-myc-induced apoptosis, and to allow c-myc to exert its oncogenic functions (Vaux et al. 1988; Brito-Babapulle et al. 1991; Bissonnette et al. 1992; Fanidi et al. 1992; Karsan et al. 1993; Mohammad et al. 1993; Oltvai et al. 1993; Marin et al. 1995). In this way bcl-2 might cooperate with c-myc in the development of BL (Fig. 5).
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PMID:Role of Epstein-Barr virus in Burkitt's lymphoma. 1144 59

Telomerase activity (TA) is increased in human cancers and cell lines and is thought to contribute to their immortality. High TA has been found to correlate with aggressive tumor behavior. The aim of this study was to determine whether increased TA in colorectal carcinoma (CRC) correlates with survival. Formalin-fixed and paraffin-embedded tissue sections from 82 CRC and 6 cases of benign colon with diverticulosis were immunohistochemically stained for telomerase reverse transcriptase (TRT) using the immunoperoxidase method. The percentage of positive nuclei was determined for each case. Survival analysis was performed using the Kaplan-Meier method. TRT immunoreactivity was always nuclear. In normal colonic mucosa, TRT immunoreactivity was detected in the bottom of crypts. However, in normal colon adjacent to CRC, telomerase immunoreactivity was detected throughout the length of the crypts, including the upper third, and frequently in the surface epithelium. Telomerase immunoreactivity in more than 25% of the cancer cell nuclei was associated with significantly poorer patient survival (P = 0.0081). We conclude that increased TA in CRC, as demonstrated by TRT immunostaining, is associated with poorer survival, and that TA is present in normal colonic mucosa and is increased in colonic mucosa near CRC. Additional studies with larger patient samples and multivariate analysis are needed to determine whether TRT expression is an independent prognostic factor in CRC.
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PMID:Immunohistochemical detection of telomerase reverse transcriptase in colorectal adenocarcinoma and benign colonic mucosa. 1219 19

Transcriptional activator proteins recruit the RNA polymerase II machinery and chromatin-modifying activities to promoters. Biochemical experiments indicate that activator proteins can associate with a large number of proteins, and many such proteins have been proposed to be direct targets of activators. However, there is great uncertainty about which biochemical interactions are physiologically relevant. Here, we develop a formaldehyde-based cross-linking procedure to identify protein-protein interactions that occur under physiological conditions. We show that the VP16 activation domain directly interacts with TATA-binding protein (TBP), TFIIB, and the SAGA histone acetylase complex in vivo.
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PMID:The VP16 activation domain interacts with multiple transcriptional components as determined by protein-protein cross-linking in vivo. 1229 14

The multisubunit Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase) complex is required to activate transcription of a subset of RNA polymerase II-dependent genes. However, the contribution of each SAGA component to transcription activation is relatively unknown. Here, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation assay, we have systematically analyzed the role of SAGA components in the recruitment of TATA-box binding protein (TBP) to SAGA-dependent promoters. We show that recruitment of TBP is diminished at a number of SAGA-dependent promoters in ada1delta, spt7delta, and spt20delta null mutants, consistent with previous biochemical data suggesting that these components maintain the integrity of the SAGA complex. We also find that Spt3p is generally required for TBP binding to SAGA-dependent promoters, consistent with biochemical and genetic experiments, suggesting that Spt3p interacts with and recruits TBP to the core promoter. By contrast, Spt8p, which has been proposed to be required for the interaction between Spt3p and TBP, is required for TBP binding at only a subset of SAGA-dependent promoters. Ada2p and Ada3p are both required for TBP recruitment to Gcn5p-dependent promoters, supporting previous biochemical data that Ada2p and Ada3p are required for the histone acetyltransferase activity of Gcn5p. Finally, our results suggest that TBP-associated-factor components of SAGA are differentially required for TBP binding to SAGA-dependent promoters. In summary, we show that SAGA-dependent promoters require different combinations of SAGA components for TBP recruitment, revealing a complex combinatorial network for transcription activation in vivo.
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PMID:Differential requirement of SAGA components for recruitment of TATA-box-binding protein to promoters in vivo. 1237 Feb 84

Doxorubicin (DOX), daunorubicin (DRB), epidoxorubicin (EDOX) and their analogues with a 3'-NH2 group in daunosamine form a covalent bond with a 2-NH2 group of guanine via a methylene group from formaldehyde (CH2O). It is assumed that a Schiff base type intermediate is formed between CH2O and the 3'-NH2 group in the reaction. This reaction is supposed to occur in the cell. New analogues of anthracyclines with formamidine functionality bound to C-3' of daunosamine and containing the bulky morpholine (DRBM, DOXM and EDOXM) or hexamethyleneimine rings attached are studied in our laboratory. These substituents decrease the association of the drugs to DNA and potentially hinder the formation of Schiff base-intermediates. Our experiments indicate that the formation of the covalent complexes by DRB, DOX and EDOX under these conditions is confirmed by a high enhancement (17-40x) of the inhibition of overall RNA synthesis by E. coli RNA polymerase on T7 DNA. DRBM and DOXM exhibit a lower enhancement of the inhibition by CH2O (7-13x). The other analogues show a 1.6-3x increase of inhibition. Hence, their covalent binding is lower than that of the parent compounds. These conclusions are confirmed by spectrophotometric estimations following removal of non-covalently associated drugs. Electrophoretic analysis of drug-DNA complexes formed in the presence of CH2O indicates that DRBM and DOXM as their parent compounds induce labile cross-links in DNA. Comparison of the results obtained at the subcellular level with cytotoxicity estimations indicates that there is a correlation between cytotoxicity of the anthracyclines on L1210 cells and transcriptional template activity of drug-DNA complexes formed in the presence of CH2O (r = 0.64; n = 9). These data confirm a notion that covalent attachment of anthracyclines to DNA is an essential event leading to cytotoxicity.
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PMID:Interactions of novel morpholine and hexamethylene derivatives of anthracycline antibiotics with DNA. 1554 Jun 9

The interaction of RNA polymerases from Escherichia coli and Thermus aquaticus with lacUV5 promoter was studied at various temperatures. Using DNA-protein cross-linking induced by formaldehyde, it was demonstrated that each RNA polymerase formed a unique pattern of contacts with DNA in the open promoter complex. In the case of E. coli RNA polymerase, beta and sigma subunits were involved into formation of cross-links with the promoter, whereas in the case of T. aquaticus RNA polymerase its beta subunit formed the cross-links with the promoter. A cross-linking pattern in promoter complexes of a hybrid holoenzyme comprised of the core-enzyme of E. coli and sigma subunit of T. aquaticus was similar to that of the E. coli holoenzyme. This suggests that DNA-protein contacts in the promoter complex are primarily determined by the core-enzyme of RNA polymerase. However, temperature-dependent behavior of contact formation is determined by the sigma subunit. Results of the present study indicate that the method of formaldehyde cross-linking can be employed for elucidation of differences in the structure of promoter complexes of RNA polymerases from various bacteria.
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PMID:Differences in contacts of RNA polymerases from Escherichia coli and Thermus aquaticus with lacUV5 promoter are determined by core-enzyme of RNA polymerase. 1633 81

Previous biochemical studies have demonstrated that Lys-123 ubiquitination of histone H2B is globally required for up-regulation of mono-, di, and trimethylation of Lys-4 of histone H3. However, recent studies have implicated H2B-Lys-123 ubiquitination in the regulation of di- and trimethylation, but not monomethylation, of H3-Lys-4 in vivo. Using a formaldehyde-based cross-linking and chromatin immunoprecipitation assay, we show that H3-Lys-4 trimethylation, but not dimethylation, is up-regulated by H2B-Lys-123 ubiquitination in vivo at the coding sequences of a set of transcriptionally active genes such as ADH1, PHO84, and PYK1. Both the ubiquitination of H2B-Lys-123 and the methylation of H3-Lys-4 are dispensable for recruitment of RNA polymerase II to the coding sequences of these genes, and hence, their transcription is not altered in the absence of these covalent modifications. However, recruitment of RNA polymerase II to the coding sequence of a galactose-inducible gene, GAL1, is significantly reduced in the absence of H2B-Lys-123 ubiquitination but not H3-Lys-4 methylation. Consistently, transcription of GAL1 is altered in the H2B-K123R point mutant strain. Finally, we show that H3-Lys-4 methylation does not regulate H3-Lys-9/14 acetylation. Collectively, our data reveal a "trans-tail" regulation of H3-Lys-4 tri- but not dimethylation by H2B-Lys-123 ubiquitination, and these modifications are dispensable for transcription of a certain set of genes in vivo.
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PMID:Functional analysis of H2B-Lys-123 ubiquitination in regulation of H3-Lys-4 methylation and recruitment of RNA polymerase II at the coding sequences of several active genes in vivo. 1667 45


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