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)

SII was purified from calf thymus tissue to apparent homogeneity by a rapid procedure. The 38-kDa protein stimulated RNA synthesis by purified calf thymus RNA polymerase II 4-fold. The calf thymus SII had similar chromatographic properties and molecular size and cross-reacted immunologically with antibodies to mouse SII (Sekimizu, K., Nakanishi, Y., Mizuno, D., and Natori, S. (1979) Biochemistry 18, 1582-1588). We have substituted the purified calf thymus SII for the partially purified HeLa transcription factor IIS fraction in a HeLa (human) transcription system reconstituted with purified factors and RNA polymerase II. The purified protein stimulated specific transcription from the adenovirus 2 major late promoter by increasing the efficiency of the elongation reaction.
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PMID:Purification and functional characterization of transcription factor SII from calf thymus. Role in RNA polymerase II elongation. 355 92

We have used affinity chromatography on columns containing immobilized calf thymus RNA polymerase II to isolate three phosphoproteins (RAP72, RAP38, and RAP30) that bind directly to RNA polymerase II. All could be isolated from cell nuclei, and all three could be detected in mouse and human tissue culture cell lines, but only RAP38 and RAP30 have so far been isolated from calf thymus. RAP38 stimulates nonspecific transcription of native DNA templates by RNA polymerase II in the presence of Mn2+; it appears to be similar or identical to SII, a previously identified RNA polymerase II stimulatory factor (Nakanishi, Y., Mitsuhashi, Y., Sekimizu, K., Yokoi, H., Tanaka, Y., Horikoshi, M., and Natori, S. (1981) FEBS Lett. 130, 69-72). Unlike RAP38, RAP72 and RAP30 do not affect nonspecific transcription by RNA polymerase II. However, RAP30 may have a role in regulating some alterations of transcription that accompany cellular differentiation; RAP30 is partially dephosphorylated when murine erythroleukemia cells are induced with dimethyl sulfoxide to undergo terminal erythroid differentiation. We suggest that phosphate groups in RNA polymerase II-binding proteins may regulate transcription by modulating the interaction of RNA polymerase II with other regulatory proteins that possess sequence recognition specificity.
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PMID:Isolation of three proteins that bind to mammalian RNA polymerase II. 386 May 4

Obstacles incurred by RNA polymerase II during primary transcript synthesis have been identified in vivo and in vitro. Transcription past these impediments requires SII, an RNA polymerase II-binding protein. SII also activates a nuclease in arrested elongation complexes and this nascent RNA shortening precedes transcriptional readthrough. Here we show that in the presence of SII and nucleotides, transcript cleavage is detected during SII-dependent elongation but not during SII-independent transcription. Thus, under typical transcription conditions, SII is necessary but insufficient to activate RNA cleavage. RNA cleavage could serve to move RNA polymerase II away from the transcriptional impediment and/or permit RNA polymerase II multiple attempts at RNA elongation. By mapping the positions of the 3'-ends of RNAs and the elongation complex on DNA, we demonstrate that upstream movement of RNA polymerase II is not required for limited RNA shortening (seven to nine nucleotides) and reactivation of an arrested complex. Arrested complexes become elongation competent after removal of no more than nine nucleotides from the nascent RNA's 3'-end. Further cleavage of nascent RNA, however, does result in "backward" translocation of the enzyme. We also show that one round of RNA cleavage is insufficient for full readthrough at an arrest site, consistent with a previously suggested mechanism of SII action.
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PMID:Nascent RNA cleavage by arrested RNA polymerase II does not require upstream translocation of the elongation complex on DNA. 750 82

There are indications that different concentrations of Sarkosyl can block transcription initiation by RNA polymerase II in vitro at different functional steps [Hawley and Roeder (1985) J. Biol. Chem. 260, 8163-8172]. Consequently, this reagent could be a very useful tool for mechanistic studies. So far, however, evidence for the selectivity of Sarkosyl effects on RNA polymerase II transcription has been only indirect. To directly investigate the effect of Sarkosyl on transcription initiation and reinitiation by RNA polymerase II, we employed the reinitiation assay based on utilization of templates containing G-free cassettes (colliding polymerases reinitiation assay, or CoPRA). These experiments showed unambiguously that, under the appropriate conditions, Sarkosyl can be used to block transcription reinitiation by RNA polymerase II while allowing a first round of initiations from preassembled initiation complexes. This inhibition is not due to a disruption of the SII-dependent elongation of the reinitiated transcripts, and the levels of Sarkosyl that prevent transcription reinitiation coincide with the levels that block preinitiation complex assembly. However, Sarkosyl addition to transcription reactions reconstituted with partially purified transcription factors was found to have several undesirable side effects. The usefulness and limitations of the Sarkosyl-based and CoPRA assays for measurements of transcription reinitiation are discussed.
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PMID:Sarkosyl block of transcription reinitiation by RNA polymerase II as visualized by the colliding polymerases reinitiation assay. 752 6

Ternary complexes of vaccinia virus RNA polymerase containing 3'-OMeGMP-arrested transcripts were purified by native gel electrophoresis. These complexes resumed elongation in situ when gel slices were incubated with magnesium and NTPs. Elongation occurred in the absence of pyrophosphate, suggesting that the blocking 3'-OMeGMP residue was removed via a novel pathway. We show that purified elongation complexes contain an intrinsic nuclease activity that shortens nascent RNA from the 3'-end. RNA cleavage was absolutely dependent on a divalent cation and was stimulated by CTP. The initial 5' cleavage product remained associated with the ternary complex and could be elongated in the presence of NTPs. Multiple stepwise cleavages generated progressively shorter chains. Purified ternary complexes containing 3'-OH-terminated RNAs also displayed nuclease activity. Involvement of the vaccinia RNA polymerase subunit rpo30 in the transcript-shortening reaction is suggested based on sequence similarity of rpo30 to mammalian protein SII (TFIIS), an extrinsic transcription factor required for nascent RNA cleavage by RNA polymerase II (Reines, D. (1991) J. Biol. Chem. 267, 3795-3800).
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PMID:Nascent RNA cleavage by purified ternary complexes of vaccinia RNA polymerase. 767 16

The rate of RNA elongation by RNA polymerase II (pol II) is affected by DNA sequences called intrinsic arrest sites. Efficient transcription through these sites requires elongation factor SII. In addition to the sequence-specific features of the DNA, we show that the acquisition of SII-dependence is a function of its "dwell-time" at an arrest site. This temperature-dependent decay in elongation potential appears irreversible, implying that factor-dependent and factor-independent elongation complexes are not mutually interconvertible at this position. TFIIF and NH4Cl are known to increase the elongation rate of pol II. Both agents preempt arrest, consistent with the idea that elongation dwell time influences the process. TFIIF and SII act upon different steps in a complementary way to prevent or resolve arrest, respectively. They are probably instrumental in facilitating the efficient transcription of large eukaryotic genes in vivo.
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PMID:Identification of a decay in transcription potential that results in elongation factor dependence of RNA polymerase II. 774 57

In the presence of elongation factor SII, arrested RNA polymerase II ternary complexes cleave 7-17 nucleotides from the 3'-ends of their nascent RNAs. It has been shown that transcription of linear templates generates apparent run-off RNAs, which are nevertheless truncated upon incubation with SII. By using high resolution gels, we demonstrate that transcription of blunt or 3'-overhung templates with RNA polymerase II generates two populations of ternary complexes. The first class pauses 5-10 bases prior to the end of the template strand. These complexes respond to SII by cleaving approximately 9-17 nucleotide RNAs from their 3'-ends and therefore may be termed arrested. A second class of complexes, which fail to respond to SII, transcribe to within 3 bases of the end of the template strand. These complexes appear to have run off the template since they have released their nascent RNAs. Run-off transcription occurs on all types of templates, but it is the predominant reaction on DNAs with 5'-overhung ends. Thus, RNA polymerase II ternary complexes that retain 5-10 bases of contact with the template strand down-stream of the catalytic site become arrested. Further reduction of downstream template contacts can lead to termination. We also show that the addition of Sarkosyl to the elongation reactions significantly changes the pattern of transcriptional arrest near the end of linear templates.
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PMID:RNA polymerase II ternary complexes may become arrested after transcribing to within 10 bases of the end of linear templates. 783 62

Transcription elongation factors stimulate the activity of DNA-dependent RNA polymerases by increasing the overall elongation rate and the completion of RNA chains. One group of such factors, which includes Escherichia coli GreA, GreB and eukaryotic SII (TFIIS), acts by inducing hydrolytic cleavage of the transcript within the RNA polymerase, followed by release of the 3'-terminal fragment. Here we report the crystal structure of GreA at 2.2 A resolution. The structure contains an amino-terminal domain consisting of an antiparallel alpha-helical coiled-coil dimer which extends into solution, reminiscent of the coiled coil in seryl-tRNA synthetases. A site near the tip of the coiled-coil 'finger' plays a direct role in the transcript cleavage reaction by contacting the 3'-end of the transcript. The structure exhibits an unusual asymmetric charge distribution which indicates the manner in which GreA interacts with the RNA polymerase elongation complex.
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PMID:Crystal structure of the GreA transcript cleavage factor from Escherichia coli. 785 24

The nucleotide sequence of a 55098 bp region from the right end of the genome of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) has been determined. Translation of the sequence identified 67 major open reading frames (ORFs) which are closely spaced and read from both DNA strands. At six positions intergenic tandem repeat arrays are found. Comparison of the predicted amino acid sequences of encoded proteins with protein sequence databases identified a number of homologies. These include three subunits of RNA polymerase, a protein with homology to transcription factor SII (TFSII), a DNA ligase, two subunits of mRNA capping enzyme, a DNA topoisomerase type II, a dUTPase, a protein kinase, three helicases, a ubiquitin-conjugating enzyme, a protein with homology to the nif S and nif S-like proteins identified in some bacteria and Saccharomyces cerevisiae, a protein with homology to both a myeloid differentiation primary response antigen (MyD116) and to a herpes simplex virus-encoded neurovirulence-associated protein (ICP34.5), a protein with homology to the ASFV-encoded structural protein p22, two proteins with homology to copies of the ASFV-encoded multigene family 360 and one protein with homology to the ASFV-encoded multigene family 110. Four genes encode proteins which have homology to each other and constitute a new multigene family (MGF100). Nine ORFs encode proteins which contain predicted transmembrane domains. The possible functions of these predicted ASFV-encoded proteins are discussed and the evolutionary relationship of ASFV to other viruses are considered. Despite the similarities in genome structure and replication strategy of ASFV with poxviruses, sequence similarity between them is low and the organization of ASFV-encoded genes is not colinear with that of the orthopoxviruses.
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PMID:Nucleotide sequence of a 55 kbp region from the right end of the genome of a pathogenic African swine fever virus isolate (Malawi LIL20/1). 802 96

RNA polymerase II may become arrested during transcript elongation, in which case the ternary complex remains intact but further RNA synthesis is blocked. To relieve arrest, the nascent transcript must be cleaved from the 3' end. RNAs of 7-17 nt are liberated and transcription continues from the newly exposed 3' end. Factor SII increases elongation efficiency by strongly stimulating the transcript cleavage reaction. We show here that arrest relief can also occur by the addition of pyrophosphate. This generates the same set of cleavage products as factor SII, but the fragments produced with pyrophosphate have 5'-triphosphate termini. Thus, the active site of RNA polymerase II, in the presence of pyrophosphate, appears to be capable of cleaving phosphodiester linkages as far as 17 nt upstream of the original site of polymerization, leaving the ternary complex intact and transcriptionally active.
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PMID:The active site of RNA polymerase II participates in transcript cleavage within arrested ternary complexes. 805 56


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