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)

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

RNA polymerase II encounters various obstacles to transcript elongation both in vivo and in vitro. These include DNA sequence elements and protein bound to the major groove of DNA. Elongation factor SII binds to RNA polymerase II and enables the enzyme to bypass these impediments. SII also activates nascent RNA cleavage by the arrested transcription elongation complex, an activity intimately involved in the readthrough process. Here we identify another type of reversible blockage to RNA polymerase II transcription, the antitumor antibiotic distamycin, which binds in the minor groove of A + T-rich DNA. SII facilitates readthrough of arrest sites resulting from DNA-binding of the drug. In response to SII, these complexes cleave their nascent RNA chains. These findings confirm that SII is a general elongation factor that potentiates transcription through a variety of impediments. They also strengthen the idea that SII stimulates transcription by activating nascent RNA cleavage. In some cases, distamycin can potentiate transcription through a naturally occurring pause site. We also show that the template undergoes a conformational change in the presence of distamycin. This suggests that distamycin can transform DNA from an elongation-non-permissive configuration into an elongation-permissive form and we take this as independent evidence confirming that DNA structure influences transcription elongation by RNA polymerase II.
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PMID:A DNA minor groove-binding ligand both potentiates and arrests transcription by RNA polymerase II. Elongation factor SII enables readthrough at arrest sites. 811 90

Previous studies have revealed that the in vitro synthesis of reinitiated transcripts by RNA polymerase II requires an additional activity, designated reinitiation transcription factor (RTF), which is distinct from all of the general class II initiation factors. While further characterizing this activity, it was found that RTF displays properties indistinguishable from those of the RNA polymerase II elongation factor SII. In addition, Western blot analysis using SII-specific antibodies revealed that human SII is a major component in purified RTF preparations. The functional equivalence of the two proteins was established using recombinant SII, which proved fully capable of substituting for RTF in the reinitiation assay. In these reconstituted reactions, transcription complexes resulting from reinitiation events required SII to proceed through a 400 bp G-free cassette, while complexes resulting from the first round of initiations were SII-independent. Reinitiations can take place in the absence of SII; however, addition of the elongation factor is essential for full extension of the reinitiated transcripts. These results suggest that events taking place at the promoter (e.g. first-round initiations versus reinitiations) can create marked differences in the properties of RNA polymerase II elongation complexes.
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PMID:Synthesis of reinitiated transcripts by mammalian RNA polymerase II is controlled by elongation factor SII. 822 77

RNA chain elongation by RNA polymerase is a dynamic process. Techniques that allow the isolation of active elongation complexes have enabled investigators to describe individual steps in the polymerization of RNA chains. This article will describe recent studies of elongation by RNA polymerase II (pol II). At least four types of blockage to chain elongation can be overcome by elongation factor SII: (a) naturally occurring "arrest" sequences, (b) DNA-bound protein, (c) drugs bound in the DNA minor groove, and (d) chain-terminating substrates incorporated into the RNA chain. SII binds to RNA polymerase II and stimulates a ribonuclease activity that shortens nascent transcripts from their 3' ends. This RNA cleavage is required for chain elongation from some template positions. As a result, the pol II elongation complex can repeatedly shorten and reextend the nascent RNA chain in a process we refer to as cleavage-resynthesis. Hence, assembly of large RNAs does not necessarily proceed in a direct manner. The ability to shorten and reextend nascent RNAs means that a transcription impediment through which only half the enzyme molecules can proceed per encounter, can be overcome by 99% of the molecules after six iterations of cleavage-resynthesis. Surprisingly, the boundaries of the elongation complex do not move upstream after RNA cleavage. The physico-chemical alterations in the elongation complex that accompany RNA cleavage and permit renewed chain elongation are not yet understood.
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PMID:Transcription elongation by RNA polymerase II: mechanism of SII activation. 831 68

RNA polymerase II (pol II) transcription complexes initiated from the adenovirus major late promoter can become blocked both in vitro and in vivo at a specific site within the first intron of the transcription unit. In vitro, polymerases that fail to read through the major late attenuation site remain stably bound to the template in a ternary complex that is indefinitely blocked from continuing elongation, a phenomenon referred to as "arrest." Elongation factor SII has been shown both to promote readthrough of this and other arrest sites and to stimulate a previously unknown 3' to 5' exonuclease activity of pol II. We have proposed that the two activities are related and that SII promotes readthrough by means of the enhancement of the exonuclease activity. In the experiments reported here, we have tested several features of that model. In particular, we have examined the hypothesis that SII stimulates readthrough by allowing the polymerase to undergo multiple cycles of removal and resynthesis of RNA bases preceding the attenuation site. In addition, we present experimental support for the proposal that the length of time polymerase pauses at the attenuation site is important to the efficiency of arrest. The results of these experiments are discussed in the context of the model.
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PMID:Transcriptional pausing, arrest, and readthrough at the adenovirus major late attenuation site. 831 69

Human RNA polymerase II is shown to be associated with a 3'-->5' exonuclease activity that removes nucleoside 5'-monophosphates from the 3' end of the transcripts in isolated ternary complexes. This activity is stimulated by SII, a protein that acts as a transcription elongation factor in vitro. In addition, we show that another transcription factor, TFIIF, stimulates a competing pyrophosphorolysis reaction. These findings raise interesting questions about the roles of these activities in vivo, including the possibility that this RNA polymerase may proofread the nascent transcript.
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PMID:Identification of a 3'-->5' exonuclease activity associated with human RNA polymerase II. 838 34

In eukaryotes the genetic material is contained within a coiled, protein-coated structure known as chromatin. RNA polymerases must recognize specific nucleoprotein assemblies and maintain contact with the underlying DNA duplex for many thousands of base pairs. Template-bound lac operon repressor from Escherichia coli arrests RNA polymerase II in vitro and in vivo [Kuhn, A., Bartsch, I. & Grummt, I. (1990) Nature (London) 344, 559-562; Deuschele, U., Hipskind, R. A. & Bujard, H. (1990) Science 248, 480-483]. We show that in a reconstituted transcription system, elongation factor SII enables RNA polymerase II to proceed through this blockage at high efficiency. lac repressor-arrested elongation complexes display an SII-activated transcript cleavage reaction, an activity associated with transcriptional read-through of a previously characterized region of bent DNA. This demonstrates factor-dependent transcription by RNA polymerase II through a sequence-specific DNA-binding protein. Nascent transcript cleavage may be a general mechanism by which RNA polymerase II can bypass many transcriptional impediments.
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PMID:Elongation factor SII-dependent transcription by RNA polymerase II through a sequence-specific DNA-binding protein. 844 9

RNA polymerase II ternary complex cleaves its nascent transcript in a 3'-->5' direction in the presence of elongation factor SII (Izban, M. G., and Luse, D. S. (1992) Genes & Dev. 6, 1342-1356; Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). We have characterized the cleavage products generated during the truncation process with a variety of stalled RNA polymerase II ternary complexes containing uniformly labeled transcripts. These complexes, which remain elongation competent, had stopped transcription because one nucleoside triphosphate was missing from the reaction mixture. Using a novel assay system, we demonstrate that cleavage occurs in predominantly dinucleotide increments, liberating 5'-phosphodinucleotides (pNpNs). In one instance with a particular C20 complex, the first cleavage event was equally partitioned between either a di-or trinucleotide increment with all subsequent truncations occurring by the preferred dinucleotide step. Our data indicate that both the kinetics and the exact increment of SII-facilitated transcript cleavage are influenced by transcript sequence.
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PMID:SII-facilitated transcript cleavage in RNA polymerase II complexes stalled early after initiation occurs in primarily dinucleotide increments. 850 20

Elongation factor SII is required to increase the efficiency of transcription by RNA polymerase II through intrinsic arrest sites. RNA polymerase II ternary complexes exhibit a ribonuclease activity in the presence of SII, truncating nascent transcripts in a 3'-->5' direction. We show here that transcript cleavage is an obligatory step in re-establishing the elongation competency of complexes that have become blocked in elongation at an intrinsic arrest site. SII-facilitated transcript cleavage by these arrested complexes released 7-14 nucleotide RNA fragments. In contrast, SII-facilitated transcript cleavage by elongation competent complexes, which are stalled because of the absence of a nucleoside triphosphate from the reaction mixture, occurred primarily in dinucleotide increments. We can partially recreate the arrested phenotype and the preference for the large cleavage increment by stalling ternary complexes such that the 3'-end of the transcript contains consecutive U residues, which mimics the sequence of the 3'-ends of transcripts in arrested complexes.
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PMID:The increment of SII-facilitated transcript cleavage varies dramatically between elongation competent and incompetent RNA polymerase II ternary complexes. 850 21

RNA polymerase II arrested at specific template locations can be rescued by elongation factor SII via RNA cleavage. The size of the products removed from the 3'-end of the RNA varies. The release of single nucleotides, dinucleotides, and larger oligonucleotides has been detected by different workers. Dinucleotides tend to originate from SII-independent complexes and 7-14 base products from SII-dependent complexes (Izban, M. G., and Luse, D. S. (1993) J. Biol. Chem. 268, 12874-12885). Different modes of cleavage have also been recognized for bacterial transcription complexes and are thought to represent important structural differences between functionally distinct transcription intermediates. Using an elongation complex "walking" technique, we have observed factor-independent complexes as they approach and become arrested at an arrest site. Dinucleotides or 7-9-base (large) oligonucleotides were released from SII-independent or dependent complexes, respectively. The abrupt shift between the release of dinucleotide versus larger products accompanied the change from factor-dependent to factor-independent elongation, as described by others. However, not all factor-independent complexes showed cleavage in dinucleotide intervals since oligonucleotides 2-6 bases long were also liberated from elongation-competent complexes. These were all 5'-coterminal oligonucleotides indicating that a preferred phosphodiester bond is targeted for cleavage in a series of related complexes. This is consistent with recent models postulating a large product binding site that can hold RNA chains whose size increases as a function of chain polymerization. A specific transitional complex was identified that acquired the ability to cleave in a large increment one base insertion event prior to attaining the arrested configuration.
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PMID:Variation in the size of nascent RNA cleavage products as a function of transcript length and elongation competence. 853 Apr 72


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