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:4.1.99.3 (PRE)
1,923 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report the first measurement of the magnetic circular dichroism (MCD) of the basic polypeptide antibiotic netropsin (Nt). The MCD shows that the longest wavelength absorption band of Nt is the sum of more than one component and permits a radically new interpretation of the circular dichroism of the complex which Nt forms with DNA. We conclude that Nt has no major effect on the CD and thus the helical structure of the bases of the DNA to which it is bound. Thus the ability of Nt to inhibit the function of DNA polymerase, RNA polymerase, and the photoreactivating enzyme must be mediated by factors other than a distortion of the helical structure of the bases.
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PMID:Magnetic circular dichroism of netropsin and natural circular dichroism of the netropsin-DNA complex. 56 96

The mammalian ribosomal RNA gene promoters exhibit a conserved sequence between positions +1 and +16 that shows a high degree of homology to the response element for glucocorticoids and progestins (GRE/PRE). These sequences bind specifically the glucocorticoid receptor and the progesterone receptor (PR) albeit with lower affinity than a canonical GRE/PRE. Because steroid hormones are known to affect expression of the ribosomal genes, we tested the influence of hormone receptors on the activity of the ribosomal RNA gene promoter in a cell-free transcription assay. Preparations of PR that induce transcription from the mouse mammary tumour virus (MMTV) promoter do not stimulate but slightly inhibit transcription from the ribosomal RNA gene promoter. This weak negative effect is not mediated through binding to the hypothetical GRE/PRE as a mutant promoter that does not bind receptor is equally repressed. Introduction of the functional MMTV GRE/PRE upstream of the basal ribosomal RNA gene promoter does not enhance its transcription in the presence of an active PR. Thus, RNA polymerase I transcription cannot be stimulated in vitro by cis elements and regulatory proteins that are active in RNA polymerase II transcription.
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PMID:Neither the endogenous nor a functional steroid hormone receptor binding site transactivate the ribosomal RNA gene promoter in vitro. 191 32

The effects of cy mutations on transcription initiation at the lambda PRE promoter were determined using abortive initiation analysis (McClure, 1980). In the presence of lambda cll protein, which activates PRE, three mutations in the -10 region dramatically reduce k2, the forward rate constant for the isomerization of closed to open complexes, but only slightly affect KB, the equilibrium constant for the initial recognition by RNA polymerase to form closed complexes. In contrast, five -35 region mutations caused decreases of 30 to 150 times in KB with much smaller effects on k2. In the absence of cll protein, the effects of mutations in the -10 region are qualitatively similar to those observed in the presence of cll protein, although the reductions in k2 are much less dramatic. In contrast, none of the mutants with defects in the -35 region is distinguishable from wildtype PRE in the absence of cll protein. Thus RNA polymerase may recognize different sequences in the -35 region in the absence of cll protein than in its presence.
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PMID:Differential effects of mutations on discrete steps in transcription initiation at the lambda PRE promoter. 622 64

Abortive and productive initiation assays were used to study transcription initiation at the PRE promoter of phage lambda in vitro. Two parameters were measured: k2, the rate constant for the transition between closed and open complexes; and KB, the equilibrium constant for the initial binding of RNA polymerase to promoter DNA. In the absence of cII protein (which activates PRE) the PRE promoter was extremely weak as expected, with k2 = 4.0 X 10(-4) S-1 and KB = 1.0 X 10(7) M-1. The addition of cII protein resulted in about a 15-fold increase in KB and a 40-fold increase in k2. Thus, cII activation of PRE results both in enhanced binding of RNA polymerase to DNA to form closed complexes and in an enchanced rate of isomerization of closed to open complexes. In addition, we found that open complexes formed in the presence of cII protein were at least four times as stable as those formed in its absence. This suggests that RNA polymerase and cII protein may remain in close contact even after complexes are formed.
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PMID:Role of cII protein in stimulating transcription initiation at the lambda PRE promoter. Enhanced formation and stabilization of open complexes. 622 41

Abortive initiation and run-off transcription assays were used to study the effects of cy mutations on activation of the phage lambda PRE promoter by cII gene product. Six point mutations in the repeated T-T-G-C sequences that flank the -35 consensus region of PRE decreased the apparent affinity of the promoter for cII protein by factors of 4-16 relative to the wild-type affinity. Kinetic analyses of transcription initiation in the presence and absence of cII protein demonstrated that five of the six mutations did not significantly affect the intrinsic interaction of RNA polymerase with PRE. Thus, these mutations differ from other cy mutations, including those in the -35 consensus region, which affect the formation of polymerase-PRE closed complexes or the isomerization of closed complexes to open complexes but do not affect the binding of cII protein. A sixth T-T-G-C mutation, cy3001, may affect intrinsic initiation by RNA polymerase as well as cII binding.
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PMID:Kinetic analysis of mutations affecting the cII activation site at the PRE promoter of bacteriophage lambda. 623 32

A current model for transcription-coupled DNA repair is that RNA polymerase, arrested at a DNA lesion, directs the repair machinery to the transcribed strand of an active gene. To help elucidate this role of RNA polymerase, we constructed DNA templates containing the major late promoter of adenovirus and a cyclobutane pyrimidine dimer (CPD) at a specific site. CPDs, the predominant DNA lesions formed by ultraviolet radiation, are good substrates for transcription-coupled repair. A CPD located on the transcribed strand of the template was a strong block to polymerase movement, whereas a CPD located on the nontranscribed strand had no effect on transcription. Furthermore, the arrested polymerase shielded the CPD from recognition by photolyase, a bacterial DNA repair protein. Transcription elongation factor SII (also called TFIIS) facilitates read-through of a variety of transcriptional pause sites by a process in which RNA polymerase II cleaves the nascent transcript before elongation resumes. We show that SII induces nascent transcript cleavage by RNA polymerase II stalled at a CPD. However, this cleavage does not remove the arrested polymerase from the site of the DNA lesion, nor does it facilitate translesional bypass by the polymerase. The arrested ternary complex is stable and competent to resume elongation, demonstrating that neither the polymerase nor the RNA product dissociates from the DNA template.
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PMID:Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimer in the DNA template. 807 11

DNA-damage formation and repair are coupled to the structure and accessibility of DNA in chromatin. DNA damage may compromise protein binding, thereby affecting function. We have studied the effect of TATA-binding protein (TBP) on damage formation by ultraviolet light and on DNA repair by photolyase and nucleotide excision repair in yeast and in vitro. In vivo, selective and enhanced formation of (6-4)-photoproducts (6-4PPs) was found within the TATA boxes of the active SNR6 and GAL10 genes, engaged in transcription initiation by RNA polymerase III and RNA polymerase II, respectively. Cyclobutane pyrimidine dimers (CPDs) were generated at the edge and outside of the TATA boxes, and in the inactive promoters. The same selective and enhanced 6-4PP formation was observed in a TBP-TATA complex in vitro at sites where crystal structures revealed bent DNA. We conclude that similar DNA distortions occur in vivo when TBP is part of the initiation complexes. Repair analysis by photolyase revealed inhibition of CPD repair at the edge of the TATA box in the active SNR6 promoter in vitro, but not in the GAL10 TATA box or in the inactive SNR6 promoter. Nucleotide excision repair was not inhibited, but preferentially repaired the 6-4PPs. We conclude that TBP can remain bound to damaged promoters and that nucleotide excision repair is the predominant pathway to remove UV damage in active TATA boxes.
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PMID:TATA-binding protein promotes the selective formation of UV-induced (6-4)-photoproducts and modulates DNA repair in the TATA box. 988 99

We have characterized the properties of immunopurified transcription complexes arrested at a specifically located cyclobutane pyrimidine dimer (CPD) using enzymatic probes and an in vitro transcription system with purified RNA polymerase II (RNAP II) and initiation factors. To help understand how RNAP II distinguishes between a natural impediment and a lesion in the DNA to initiate a repair event, we have compared the conformation of RNAP II complexes arrested at a CPD with complexes arrested at a naturally occurring elongation impediment. The footprint of RNAP II arrested at a CPD, using exonuclease III and T4 DNA polymerase's 3'-->5' exonuclease, covers approximately 35 base pairs and is asymmetrically located around the dimer. A similar footprint is observed when RNAP II is arrested at the human histone H3.3 arrest site. Addition of elongation factor SII to RNAP II arrested at a CPD produced shortened transcripts of discrete lengths up to 25 nucleotides shorter than those seen without SII. After addition of photolyase and exposure to visible light, some of the transcripts could be reelongated beyond the dimer, suggesting that SII-mediated transcript cleavage accompanied significant RNAP II backup, thereby providing access of the repair enzyme to the arresting CPD.
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PMID:Structural characterization of RNA polymerase II complexes arrested by a cyclobutane pyrimidine dimer in the transcribed strand of template DNA. 1044 84

DNA repair by photolyase (photoreactivation) and nucleotide excision repair (NER) are the major pathways to remove UV-induced cyclobutane-pyrimidine dimers (CPDs). The nucleolus is a nuclear subcompartment containing the ribosomal RNA genes (rDNA) of which a fraction is transcribed by RNA polymerase I (RNAP-I), and the rest is silenced. Here yeast was used to investigate how photoreactivation and NER contribute to repair of active and inactive rDNA. Cells were irradiated with UV light and exposed to different repair conditions. Nuclei were isolated, and the active genes were separated from the inactive genes by restriction endonuclease digestion. CPDs were measured in total rDNA, in both fractions, and in the GAL10 gene. Repair in rDNA was as efficient as in GAL10 indicating that both pathways have unrestricted access to the nucleolus. Photoreactivation was much faster than NER and therefore was the predominant repair pathway. Active genes were faster repaired by photolyase than were silenced genes providing evidence for an open chromatin structure during repair. The transcribed strands of active genes, but not of inactive genes, were slightly faster repaired by NER providing evidence for transcription-coupled repair by RNAP-I. There was no pronounced inhibition of photoreactivation by RNAP-I in the transcribed strand, which is in contrast to genes transcribed by RNAP-II and suggests different stabilities of RNAP-I and RNAP-II stalled at CPDs.
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PMID:Repair of active and silenced rDNA in yeast: the contributions of photolyase and transcription-couples nucleotide excision repair. 1180 5

UV photofootprinting and repair of pyrimidine dimers by photolyase was used to investigate chromatin structure, protein-DNA interactions, and DNA repair in the spacer and promoter of Saccharomyces cerevisiae rRNA genes. Saccharomyces cerevisiae contains about 150 copies of rRNA genes separated by nontranscribed spacers. Under exponential growth conditions about half of the genes are transcribed by RNA polymerase I (RNAP-I). Initiation of transcription requires the assembly of the upstream activating factor (UAF), the core factor (CF), TATA binding protein, and RNAP-I with Rrn3p on the upstream element and core promoter. We show that UV irradiation of wild-type cells and transcription factor mutants generates photofootprints in the promoter elements. The core footprint depends on UAF, while the UAF footprint was also detected in absence of the CFs. Fractionation of active and inactive promoters showed the core footprint mainly in the active fraction and similar UAF footprints in both fractions. DNA repair by photolyase was strongly inhibited in active promoters but efficient in inactive promoters. The data suggest that UAF is present in vivo in active and inactive promoters and that recruitment of CF and RNAP-I to active promoters generates a stable complex which inhibits repair.
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PMID:RNA polymerase I transcription factors in active yeast rRNA gene promoters enhance UV damage formation and inhibit repair. 1571 19


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