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)

TFIIS is a general transcription elongation factor that helps arrested RNA polymerase II elongation complexes resume transcription. We have previously shown that yeast TFIIS (yTFIIS) comprises three structural domains (I-III). The three-dimensional structures of domain II and part of domain III have been previously reported, but neither domain can autonomously stimulate transcription elongation. Here we report the NMR structural analysis of residues 131-309 of yTFIIS which retains full activity and contains all of domains II and III. We confirm that the structure of domain II in the context of fully active yTFIIS is the same as that determined previously for a shorter construct. We have determined the structure of the C-terminal zinc ribbon domain of active yTFIIS and shown that it is similar to that reported for a shorter construct of human TFIIS. The region linking domain II with the zinc ribbon of domain III appears to be conformationally flexible and does not adopt a single defined tertiary structure. NMR analysis of inactive mutants of yTFIIS support a role for the linker region in interactions with the transcription elongation complex.
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PMID:Yeast transcript elongation factor (TFIIS), structure and function. I: NMR structural analysis of the minimal transcriptionally active region. 971 87

The transcriptionally active fragment of the yeast RNA polymerase II transcription elongation factor, TFIIS, comprises a three-helix bundle and a zinc ribbon motif joined by a linker region. We have probed the function of this fragment of TFIIS using structure-guided mutagenesis. The helix bundle domain binds RNA polymerase II with the same affinity as does the full-length TFIIS, and this interaction is mediated by a basic patch on the outer face of the third helix. TFIIS mutants that were unable to bind RNA polymerase II were inactive for transcription activity, confirming the central role of polymerase binding in the TFIIS mechanism of action. The linker and zinc ribbon regions play roles in promoting cleavage of the nascent transcript and read-through past the block to elongation. Mutation of three aromatic residues in the zinc ribbon domain (Phe269, Phe296, and Phe308) impaired both transcript cleavage and read-through. Mutations introduced in the linker region between residues 240 and 245 and between 250 and 255 also severely impaired both transcript cleavage and read-through activities. Our analysis suggests that the linker region of TFIIS probably adopts a critical structure in the context of the elongation complex.
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PMID:Yeast transcript elongation factor (TFIIS), structure and function. II: RNA polymerase binding, transcript cleavage, and read-through. 971 88

The transcriptional factor TFIIS helps overcome elongation barriers and enhances proofreading by RNA polymerase II. These TFIIS functions may be modulated by the TFIIS zinc ribbon domain through interactions with nucleic acids in the elongation complex. Within this zinc ribbon domain, the dipeptide sequences Asp261-Glu262 and Arg276-Trp277 have been shown to be critical for its function by mutant analysis. The sequence Asp261-Glu262 has been suggested to participate in metal binding within the RNA polymerase II active site. We now show that the sequence Arg276-Trp277 interacts with nucleic acids through a combination of electrostatic and stacking interactions. The interaction of the indole side chain of the tryptophan residue with nucleic acid bases is demonstrated by a characteristic and reversible decrease in the zinc ribbon fluorescence intensity as a function of oligonucleotide concentration. These interactions are salt sensitive (maximum interaction at 200 mM and no interaction at 500 mM NaCl), suggesting that the tryptophan stacking with nucleic acid base accompanies electrostatic contacts. The oligonucleotide-zinc ribbon interactions exhibit small but significant base preferences, as shown by the dependence of Keq on base composition, with decreasing Keq in the order U > T > A > C >> G. Within the variety of homopolymeric single- and double-stranded deoxy- and ribooligonucleotides, the oligonucleotide rU12-18.dA20 exhibited a 2-6-fold binding preference relative to other oligonucleotides. This preferential binding of the zinc ribbon to sequences composed of rU.dA base pairs, which are generally associated with elongation blocks, may help in overcoming elongation barriers. Since the mRNA proofreading and enhancement of elongation involve cleavage of ribonucleotide of the mismatched pair and the weakly paired rU.dA nucleotides, but not the stably paired rC.dG nucleotides, we propose that the Arg276-Trp277 sequence in the TFIIS zinc ribbon may serve as a scanner connected to the transcript cleavage apparatus for weakly paired or mismatched nucleotides by employing indole ring stacking with the bases as a criterion of determining their subsequent removal. The striking similarity in preference for mismatched and weakly paired nucleotides for binding and for excision suggests a functional relationship between binding and cleavage reactions.
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PMID:Preferential interaction of the mRNA proofreading factor TFIIS zinc ribbon with rU.dA base pairs correlates with its function. 972 22

Budding yeast RNA polymerase III (Pol III) contains a small, essential subunit, named C11, that is conserved in humans and shows a strong homology to TFIIS. A mutant Pol III, heterocomplemented with Schizosaccharomyces pombe C11, was affected in transcription termination in vivo. A purified form of the enzyme (Pol III Delta), deprived of C11 subunit, initiated properly but ignored pause sites and was defective in termination. Remarkably, Pol III Delta lacked the intrinsic RNA cleavage activity of complete Pol III. In vitro reconstitution experiments demonstrated that Pol III RNA cleavage activity is mediated by C11. Mutagenesis in C11 of two conserved residues, which are critical for the TFIIS-dependent cleavage activity of Pol II, is lethal. Immunoelectron microscopy data suggested that C11 is localized on the mobile thumb-like stalk of the polymerase. We propose that C11 allows the enzyme to switch between an RNA elongation and RNA cleavage mode and that the essential role of the Pol III RNA cleavage activity is to remove the kinetic barriers to the termination process. The integration of TFIIS function into a specific Pol III subunit may stem from the opposite requirements of Pol III and Pol II in terms of transcript length and termination efficiency.
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PMID:The RNA cleavage activity of RNA polymerase III is mediated by an essential TFIIS-like subunit and is important for transcription termination. 986 39

Activation of gene transcription in metazoans is a multistep process that is triggered by factors that recognize transcriptional enhancer sites in DNA. These factors work with co-activators to direct transcriptional initiation by the RNA polymerase II apparatus. One class of co-activator, the TAF(II) subunits of transcription factor TFIID, can serve as targets of activators and as proteins that recognize core promoter sequences necessary for transcription initiation. Transcriptional activation by enhancer-binding factors such as Sp1 requires TFIID, but the identity of other necessary cofactors has remained unknown. Here we describe a new human factor, CRSP, that is required together with the TAF(II)s for transcriptional activation by Sp1. Purification of CRSP identifies a complex of approximate relative molecular mass 700,000 (M(r) approximately 700K) that contains nine subunits with M(r) values ranging from 33K to 200K. Cloning of genes encoding CRSP subunits reveals that CRSP33 is a homologue of the yeast mediator subunit Med7, whereas CRSP150 contains a domain conserved in yeast mediator subunit Rgr1. CRSP p200 is identical to the nuclear hormone-receptor co-activator subunit TRIP2/PBP. CRSPs 34, 77 and 130 are new proteins, but the amino terminus of CRSP70 is homologous to elongation factor TFIIS. Immunodepletion studies confirm that these subunits have an essential cofactor function. The presence of common subunits in distinct cofactor complexes suggests a combinatorial mechanism of co-activator assembly during transcriptional activation.
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PMID:The transcriptional cofactor complex CRSP is required for activity of the enhancer-binding protein Sp1. 998 12

Regulation of gene expression in the domain Archaea, and specifically hyperthermophiles, has been poorly investigated so far. Biochemical experiments and genome sequencing have shown that, despite the prokaryotic cell and genome organization, basal transcriptional elements of members of the domain Archaea (i.e., TATA box-like sequences, RNA polymerase, and transcription factors TBP, TFIIB, and TFIIS) are of the eukaryotic type. However, open reading frames potentially coding for bacterium-type transcription regulation factors have been recognized in different archaeal strains. This finding raises the question of how bacterial and eukaryotic elements interact in regulating gene expression in Archaea. We have identified a gene coding for a bacterium-type transcription factor in the hyperthermophilic archaeon Sulfolobus solfataricus. The protein, named Lrs14, contains a potential helix-turn-helix motif and is related to the Lrp-AsnC family of regulators of gene expression in the class Bacteria. We show that Lrs14, expressed in Escherichia coli, is a highly thermostable DNA-binding protein. Bandshift and DNase I footprint analyses show that Lrs14 specifically binds to multiple sequences in its own promoter and that the region of binding overlaps the TATA box, suggesting that, like the E. coli Lrp, Lrs14 is autoregulated. We also show that the lrs14 transcript is accumulated in the late growth stages of S. solfataricus.
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PMID:An Lrp-like protein of the hyperthermophilic archaeon Sulfolobus solfataricus which binds to its own promoter. 1004 78

Full-length copies of cDNA of the hRPC11 gene encoding the smallest specific subunit of nuclear RNA polymerase III were identified among human transcripts with the use of the RT-PCR technique. The cloning of the first orthologue of the subunit RPC11 from a multicellular organism and the comparison of subunit hRPC11 of Homo sapiens (108 aa; M(r), 12.3 kDa; pI 8.05) deduced from the cDNA primary structure with the homologous components of RNA polymerase III from Saccharomyces cerevisiae and Schizosaccharomyces pombe revealed the most important functional domains: a Zn-binding motif of the classic type (CxxCx16-17CxxC) at the N-terminal region, and two extended regions of homology (KEVDDVLGG and RSADEPM) in the central and C-terminal parts of the molecule, respectively. The C-terminus of the RPC11 subunits is highly homologous to the unique zinc ribbon of the elongation factor TFIIS, which suggests a role for this subunit in the elongation or termination of RNA synthesis.
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PMID:[Molecular identification and characteristics of hRPC11, the smallest specific subunit of human RNA polymerase III]. 1007 44

RNA polymerase II stalled at a lesion in the transcribed strand is thought to constitute a signal for transcription-coupled repair. Transcription factors that act on RNA polymerase in elongation mode potentially influence this mode of repair. Previously, it was shown that transcription elongation factors TFIIS and Cockayne's syndrome complementation group B protein did not disrupt the ternary complex of RNA polymerase II stalled at a thymine cyclobutane dimer, nor did they enable RNA polymerase II to bypass the dimer. Here we investigated the effect of the transcription factor 2 on RNA polymerase II and RNA polymerase I stalled at thymine dimers. Transcription factor 2 is known to release transcripts from RNA polymerase II early elongation complex generated by pulse-transcription. We found that factor 2 (which is also called release factor) disrupts the ternary complex of RNA polymerase II at a thymine dimer and surprisingly exerts the same effect on RNA polymerase I. These findings show that in mammalian cells a RNA polymerase I or RNA polymerase II transcript truncated by a lesion in the template strand may be discarded unless repair is accomplished rapidly by a mechanism that does not displace stalled RNA polymerases.
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PMID:Human transcription release factor 2 dissociates RNA polymerases I and II stalled at a cyclobutane thymine dimer. 1045 50

We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem-loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.
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PMID:Assembly of the nuclear transcription and processing machinery: Cajal bodies (coiled bodies) and transcriptosomes. 1058 65

The RPB9 subunit of RNA polymerase II regulates transcription elongation activity and is required for the action of the transcription elongation factor, TFIIS. RPB9 comprises two zinc ribbon domains joined by a conserved linker region. The C-terminal zinc ribbon is similar in sequence to that found in TFIIS. To elucidate the relationship between the structure and transcription elongation function of RPB9, we initiated a mutagenesis study on the Saccharomyces cerevisiae homologue. The individual zinc ribbon domains, in isolation or in combination, could not stimulate transcription by a polymerase lacking RPB9, pol IIDelta9. Mutations in the N-terminal zinc ribbon had little effect on transcription activity. By contrast, mutations in the acidic loop that connects the second and third beta-strands of the C-terminal zinc ribbon were completely inactive for transcription. Interestingly, the analogous residues in TFIIS are also critical for elongation activity. A conserved charged stretch in the linker region (residues 89-95, DPTLPR) mediated the interaction with RNA polymerase II.
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PMID:Yeast RNA polymerase II subunit RPB9. Mapping of domains required for transcription elongation. 1064 77


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