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 transcriptional potential of the hsp70 heat shock gene promoter is established prior to induction by stress. It has been shown previously that the TBP subunit of TFIID is associated with the TATA element and that RNA polymerase II is paused downstream from the transcription start site. In order to identify new interactions involved in establishing this potentiated state, a detailed analysis of the molecular architecture of a single copy of the hsp70 promoter was performed. A suitably marked promoter was stably integrated using P-element-mediated transformation so as to overcome any ambiguity that might be associated with analyzing the five copies of the endogenous gene. Genomic footprinting using DNase I revealed two previously unidentified interactions. First, the GAGA element located at -120 is protected by protein. Secondly, the pattern of DNase I cleavage in the vicinity of the transcription start is found to bear significant similarity to the pattern associated with binding of purified TFIID. Noting that purified GAGA factor and TFIID interact similarly with the hsp70 and H3 promoters, the architecture of the endogenous H3 promoter was analyzed to determine what interactions might be needed to establish a potentiated state containing a paused polymerase. Despite the detection of TFIID and GAGA on the H3 promoter, no paused polymerase is evident. In addition, no proteins appear to interact with the transcription start. These results suggest that the GAGA factor and TFIID are not sufficient to establish a potentiated state containing paused polymerase and that TFIID interactions downstream from the TATA element could be important for pausing.
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PMID:Genomic footprinting of the hsp70 and histone H3 promoters in Drosophila embryos reveals novel protein-DNA interactions. 766 10

We have shown previously that the majority of RNA polymerase II complexes initiated at the c-myc gene are paused in the promoter-proximal region, similar to observations in the Drosophila hsp70 gene. Our analyses define the TATA box or initiator sequences in the c-myc gene as necessary components for the establishment of paused RNA polymerase II. Deletion of upstream sequences or even the TATA box does not influence significantly the degree of transcriptional initiation or pausing. Deletion of both the TATA box and sequences at the transcription initiation site, however, abolishes transcriptional pausing of transcription complexes but still allows synthesis of full-length RNA. Further analyses with synthetic promoter constructs reveal that the simple combination of upstream activator with TATA consensus sequences or initiator sequences act synergistically to recruit high levels of RNA polymerase II complexes. Only a minor fraction of these complexes escapes into regions further downstream. Several different trans-activation domains fused to GAL4-DNA-binding domains, including strong activators such as VP16, do not eliminate promoter-proximal pausing of RNA polymerase. Thus, we conclude that pausing of RNA polymerase II is a common phenomenon in eukaryotic transcription and does not require complex promoter structures. Further analyses reveal that enhancers have a modest influence on transcription initiation and on release of transcription complexes out of the pause site but may function primarily to increase the elongation competence of transcription complexes.
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PMID:Promoter-proximal pausing of RNA polymerase II defines a general rate-limiting step after transcription initiation. 769 46

GAGA protein binds specific CT.GA-rich DNA sequences in vitro, and many of these sequences are required for transcription in vivo. GAGA protein has been implicated in the transcription of numerous Drosophila genes, including hsp70, hsp26, actin 5C, and Ubx. Here, we examine the in vivo distribution of GAGA protein on a number of Drosophila genes that do and do not have CT-rich sequences by use of a UV cross-linking technique. Prior to heat shock, GAGA protein is associated with the promoter regions of the uninduced hsp70 and hsp26 genes. Upon heat shock induction, GAGA protein is recruited to their transcription units with its distribution coincident with that of RNA polymerase II. The recruitment of GAGA protein to the hsp70 gene after an instantaneous heat shock occurs in a 5' to 3' manner with kinetics similar to RNA polymerase. GAGA protein has been shown to disrupt nucleosome both in vivo and in vitro. We propose that GAGA protein may function in vivo both by binding constitutively to its high-affinity binding sites and by spreading through the induced gene opening the chromatin structure allowing polymerase to elongate efficiently.
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PMID:Distribution of GAGA protein on Drosophila genes in vivo. 774 51

The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II is essential in vivo, and is found in either an unphosphorylated (IIa) or hyperphosphorylated (IIo) form. The Drosophila uninduced hsp70 and hsp26 genes, and the constitutively expressed beta-1 tubulin and Gapdh-2 genes, contain an RNA polymerase II complex which pauses after synthesizing a short transcript. We report here that, using an in vivo ultraviolet crosslinking technique and antibodies directed against the IIa and IIo forms of the CTD, these paused polymerases have an unphosphorylated CTD. For genes containing a 5' paused polymerase, passage of the paused RNA polymerase into an elongationally competent mode in vivo coincides with phosphorylation of the CTD. Also, the level of phosphorylation of the CTD of elongating polymerases is shown not to be related to the level of transcription, but is promoter specific.
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PMID:Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation. 801 13

A protein--DNA complex containing TFIID has been analyzed by crosslinking. The TBP subunit of TFIID crosslinked to the TATA element but not to any of the regions further downstream which were tested. A 150 kd polypeptide, which corresponds in size to one of the TBP-associated factors (TAFs), crosslinked to a region between +10 and +15 and a second region between +35 and +47. Another polypeptide of greater than 205 kd (also a potential TAF) crosslinked preferentially to the region between +35 and +42. The +10 to +15 region has been recently implicated in hsp70 promoter recognition by TFIID, and the most downstream contacts overlap with the region where RNA polymerase II pauses on the hsp70 promoter in noninduced cells. Crosslinking revealed that as the salt concentration was increased, the TBP interaction was largely unaffected whereas the protein/DNA interactions downstream of the TATA element were disrupted. We propose that during the formation of a transcription complex, TATA-dependent interactions could be disrupted in the vicinity of the start site and the region immediately downstream. A protein contact downstream of +35 might function in pausing polymerase.
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PMID:Protein/DNA crosslinking of a TFIID complex reveals novel interactions downstream of the transcription start. 813 22

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) limits RNA polymerase II transcription to a gene's 5'-end. Transcription of the uninduced Drosophila hsp70 gene is likewise restricted to the 5'-end, where the polymerase resides in a paused state. Furthermore, paused elongation complexes formed on the uninduced hsp70 gene and in DRB-inhibited reactions can both be restarted by Sarkosyl or high salt. These similarities prompted us to explore whether these complexes were generated by a block at the same polymerase modification step. In vivo UV cross-linking and KMnO4 hyperreactive site mapping show that while the naturally paused polymerase is restricted to the first approximately 42 base pairs of hsp70, DRB treatment of heat-induced cells allows the polymerase to transcribe past this site. Therefore, the DRB-sensitive step is probably not rate-limiting for hsp70 transcription under uninduced conditions. DRB treatment did, however, lead to the reduction of KMnO4 hyper-reactivity on hsp70 and hsp26 in a region correlating with open polymerase and/or early elongation complexes, suggesting a site for the DRB-sensitive polymerase modification step. Finally, we used the techniques of polymerase-DNA cross-linking and KMnO4 hyper-reactive site mapping to analyze the natural polymerase termination process at the 3'-end of the hsp26 gene. The data obtained are consistent with polymerases terminating at multiple sites downstream of the polyadenylation site.
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PMID:Polymerase processivity and termination on Drosophila heat shock genes. 822 16

Regulation of transcriptional elongation is emerging as an important control mechanism for eukaryotic gene expression. In this essay, we review the basis of the current view of the regulation of elongation in the human c-myc gene and discuss similarities in elongation control among the c-myc, Drosophila hsp70 and the HIV-1 genes. Based upon these similarities, we propose a model for control of expression of these genes at the elongation phase of transcription. This model suggests that distinct promoter elements direct the assembly of RNA polymerase II transcription complexes which differ in their elongation efficiency.
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PMID:Common mechanisms for the control of eukaryotic transcriptional elongation. 827 41

The regulation of many eukaryotic genes occurs at the level of transcriptional elongation. On the uninduced hsp70 gene of Drosophila melanogaster, for example, an RNA polymerase II complex has initiated transcription but has paused early in elongation. In this study, we examine pausing on hsp70 and two of the small heat shock genes (hsp27 and hsp26) at high resolution, using a technique that utilizes paramagnetic particle-mediated selection of terminated run-on transcripts. This technique provides precise information on the distribution of RNA polymerase within each transcription unit. It also details the progression of 5' cap formation on the elongating transcripts. For each gene, we find polymerases paused over a relatively narrow promoter-proximal region. The regions are generally around 20 nucleotides wide, with two preferred pausing positions spaced roughly 10 nucleotides apart or about one turn of the helix. The bulk of capping occurs as transcripts pass between 20 and 30 nucleotides in length. Interestingly, in the three genes examined here, elongational pausing and 5' cap formation appear largely coincident.
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PMID:In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes. 836 44

We used a nuclear run-on assay as a novel approach to study the changes in transcriptional activity that take place in Drosophila melanogaster during heat shock. In response to a rapid temperature upshift, total transcriptional activity in cultured KC161 cells decreased proportionally to the severity of the shock. After extended stress at 37 degrees C (15 min or more), transcription was severely reduced, and at 39 degrees C most transcription was instantaneously arrested. However, strikingly different responses were observed for individual genes. Transcription of histone H1 genes was severely inhibited even under mild heat shock conditions. Transcription of the actin 5C gene decreased progressively with increasing temperature, while transcription of the core histone genes or of the heat shock cognate genes was repressed only under severe heat shock conditions. Transcriptional activation of the D. melanogaster heat shock genes was also investigated. In unshocked cells, hsp84 was moderately transcribed, while transcriptional activity at the other protein-coding heat shock genes was undetectable (less than 0.2 polymerases per gene). Engaged but paused RNA polymerase molecules were found at the hsp70 and hsp26 genes, but not at the other heat shock genes. The rates of transcription increased with increasing temperature with a peak of expression at around 35 degrees C. At 37 degrees C, induction was less efficient, and no induction was achieved after a rapid shift to 39 degrees C. Increased transcription of the heat shock genes was observed within 1-2 min of heat shock, and maximal rates were reached within 2-5 min. Despite very similar profiles of response, different heat shock genes were transcribed at strikingly different rates, which varied over a 20-fold range. The noncoding heat shock locus 93D was transcribed at a very high rate under non-heat shock conditions, and showed a transcriptional response to elevated temperatures different from that of protein-coding heat shock genes. An estimation of the absolute rates of transcription at different temperatures was obtained.
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PMID:Transcriptional regulation in Drosophila during heat shock: a nuclear run-on analysis. 848 75

Heat shock rapidly activates expression of some genes and represses others. The kinetics of changes in RNA polymerase distribution on heat shock-modulated genes provides a framework for evaluating the mechanisms of activation and repression of transcription. Here, using two methods, we examined the changes in RNA polymerase II association on a set of Drosophila genes at 30-s intervals following an instantaneous heat shock. In the first method, Drosophila Schneider line 2 cells were quickly frozen to halt transcription, and polymerase distribution was analyzed by a nuclear run-on assay. RNA polymerase transcription at the 5' end of the hsp70 gene could be detected within 30 to 60 s of induction, and by 120 s the first wave of polymerase could already be detected near the 3' end of the gene. A similar rapid induction was found for the small heat shock genes (hsp22, hsp23, hsp26, and hsp27). In contrast to this rapid activation, transcription of the histone H1 gene was found to be rapidly repressed, with transcription reduced by approximately 90% within 300 s of heat shock. Similar results were obtained by an in vivo UV cross-linking assay. In this second method, cell samples removed at 30-s intervals were irradiated with 40-microseconds bursts of UV light from a Xenon flash lamp, and the distribution of polymerase was examined by precipitating UV cross-linked protein-DNA complexes with an antibody to RNA polymerase II. Both approaches also showed the in vivo rate of movement of the first wave of RNA polymerase through the hsp70 gene to be approximately 1.2 kb/min.
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PMID:Rapid changes in Drosophila transcription after an instantaneous heat shock. 849 61


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