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)

We have mapped principal sites in the Escherichia coli RNA polymerase molecule that are exposed to attack by trypsin under limited proteolysis conditions. The 1342-amino acid-long beta subunit is alternatively cleaved at Arg903 or Lys909. The cleavage occurs adjacent to a dispensable domain (residues 940-1040) that is absent in the homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria. In E. coli, this region can be disrupted with genetic deletions and insertions without the loss of RNA polymerase function. Insertion of 127 amino acids into this region introduces a new highly labile site for trypsin proteolysis. The dispensable domain carries the epitope for monoclonal antibody PYN-6 (near residue 1000), which can be used for anchoring the catalytically active enzyme on a solid support. We also report the identification of a secondary trypsin cleavage at Arg81 of the beta' subunit within a putative zinc-binding domain that is conserved in prokaryotes and chloroplasts.
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PMID:Mapping of trypsin cleavage and antibody-binding sites and delineation of a dispensable domain in the beta subunit of Escherichia coli RNA polymerase. 174 64

To investigate the mechanism of action of the cowpea mosaic virus (CPMV) 24K protease, a full-length cDNA clone of bottom component (B) RNA has been constructed from which RNA can be transcribed in vitro using T7 RNA polymerase. Translation of the resulting RNA in rabbit reticulocyte lysate leads to the synthesis of a 200 kDa product (the 200K protein) which cleaves itself in a manner identical to that of the product translated from B RNA isolated from virions. Site-directed mutagenesis of the full-length clone was used to examine the effects of altering individual amino acids in the 24K protease on its activity. The results obtained are consistent with the prediction that the 24K protease is structurally similar to the trypsin-like family of serine proteases and suggest that His40, Glu76, and Cys166 comprise the active site. Substitution of Cys166 by a serine residue results in an enzyme with reduced catalytic activity.
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PMID:Mutational analysis of the putative catalytic triad of the cowpea mosaic virus 24K protease. 188 92

Transcription factor IID from Saccharomyces cerevisiae (YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0 degrees C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.
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PMID:Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change. 198 53

Gene 32 protein (g32P), the replication accessory protein from bacteriophage T4, is a zinc metalloprotein which binds with high cooperativity to single-stranded (ss) nucleic acids. The basic N-terminal 21 amino acids (termed the "B" domain) is required for highly cooperative (omega approximately 500) binding of g32P monomers to ss nucleic acids. As part of our studies to systematically evaluate the structural features of the B domain important for cooperative binding, a homogeneous source of g32P which binds noncooperatively to nucleic acids (omega = 1) and is devoid of contamination by native g32P is needed. Herein, we describe large-scale overexpression and purification of recombinant g32P lacking the tryptic N-terminal B domain (residues 1-21), designated g32P-B, as well as its physiochemical and nucleic acid binding properties. G32P-B is readily purified from the soluble fraction of Escherichia coli BL21 (DE3) transformed with the plasmid pT7g32-B.wt which contains the g32P-B coding sequences under inducible transcriptional control of T7 RNA polymerase. Anion exchange, ssDNA-cellulose and phenyl-Sepharose chromatographies give rise to highly homogeneous g32P-B, free of contaminating nucleic acid. Recombinant g32P-B has the expected N-terminal primary structure and contains stoichiometric Zn(II). It also has the expected globular structure as shown by 1H NMR spectroscopy, hydrodynamic measurements, and the ability to selectively remove the carboxyl-terminal "A" domain to form the trypsin-resistant g32P-(A + B) DNA-binding core fragment. Quantitative ss nucleic acid binding experiments of g32P-B to poly(dT) (0.05 M NaCl, pH 8.1, 20 degrees C) show that all equilibrium binding isotherms can be fit with omega = 1 and Kobs = 5.2 (+/- 1.6) x 10(5) M-1, with a moderate electrostatic component to the binding free energy, delta log Kobs/delta log[NaCl] = -3.0 +/- 0.2. Under identical solution conditions, g32P-(A + B) derived from g32P-B binds to poly(dT) noncooperatively as expected, but with an approximately 80-fold higher apparent affinity, Kobs = 4.0 (+/- 2.0) x 10(7) M-1, and detectable enhanced salt sensitivity, delta log Kobs/delta log[NaCl] = -3.9 +/- 0.3. As the salt concentration is raised, the relative difference in Kobs between the g32P-(A + B) and g32P-B is gradually reduced such that extrapolation of the log-log plots to 1 M Na+ standard state gives similar Kobs within experimental error. Qualitatively similar observations are also found upon binding to the ribohomopolymer, poly(U).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Overexpression, purification, and characterization of recombinant T4 gene 32 protein22-301 (g32P-B). 219 20

When wheat-germ RNA polymerase II is subjected to mild proteolytic attack in the presence of trypsin, the resulting form of the enzyme migrates as a single species on electrophoresis in native polyacrylamide gels, with an apparent Mr significantly smaller than that of the native enzyme. Analysis by denaturing gel electrophoresis of the truncated eukaryotic polymerase revealed that the two largest subunits of the native enzyme, i.e. the 220,000-Mr and 140,000-Mr subunits, were cleaved, giving rise to shorter polypeptide chains of Mr 172,800, 155,000, 143,000, 133,800, 125,000 and 115,000. The use of affinity-purified antibodies directed against each of the two large subunits of the native enzyme allowed us to probe for possible precursor/product relationships between the 220,000-Mr and 140,000-Mr subunits of wheat-germ RNA polymerase II and their breakdown products generated in the presence of trypsin. None of the smaller subunits of the plant RNA polymerase II appeared to be sensitive to trypsin attack. The results indicate that the truncated RNA polymerase retained a multimeric structure, and therefore that the proteolyzed largest subunits of the enzyme remained associated with the smaller ones. Furthermore, in transcription of a poly[d(A-T)] template, the catalytic activity of the proteolyzed form of wheat-germ RNA polymerase II was identical to that of the native enzyme. Therefore, the protein domains that can be deleted by the action of trypsin from the two large subunits of the plant transcriptase are not involved in DNA binding and/or nucleotide binding, and do not play an important role in template-directed catalysis of phosphodiester bond formation.
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PMID:Analysis of wheat-germ RNA polymerase II by trypsin cleavage. The integrity of the two largest subunits of the enzyme is not mandatory for basal transcriptional activity. 224 2

Two proteolytically modified forms of T7 RNA polymerase have been characterized with respect to transcription initiation and processivity. One species, denoted 80K-20K, is singly cleaved within the region of the polypeptide chain between amino acids 172 and 180. The second species, denoted 80K, is generated by extensive proteolysis of the N-terminal 20K domain by trypsin. The 80K-20K form is fully active in initiation and escape from abortive cycling. It is deficient only in processivity on long DNA templates. Likewise, the 80K species shows initiation kinetics and abortive product synthesis similar to those of the native enzyme. This latter species, however, is unable to escape abortive cycling and shows no synthesis of transcripts longer than about eight bases. Studies of RNA and DNA binding to the three different forms of the enzyme by gel retention assays reveal that the native (98K), the 80K-20K, and the 80K species all form specific complexes with promoter-containing DNA. In addition, the native enzyme binds nonspecifically to double-stranded DNA, while the 80K-20K and 80K enzymes do not. The native enzyme also binds RNA. This RNA binding is reduced in the 80K-20K enzyme and is absent in the 80K species. We suggest a model for T7 RNA polymerase wherein the 20K N-terminal domain of the protein or a shared region between the N- and the C-terminal domains of the protein forms a nonspecific polynucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Processivity of proteolytically modified forms of T7 RNA polymerase. 246 Jan 33

Polypeptides have been defined by studying structural and nonstructural proteins. The rotavirus outer capsid is made up of three proteins: VP7, VP3 and VP9. VP7 is a glycoprotein involved in cell attachment and viral maturation. VP3 is associated with hemagglutination and trypsin activation of virus infectivity; both contain type-specific neutralization determinants. A biological function has not yet been completely defined for VP9. VP6, the main protein of the inner capsid is necessary for mRNA synthesis by the viral transcriptase and determines the subgroup antigenic specificity. These two capsids surround the core which consists of three proteins VP1, VP2, and the product of segment 3, associated with RNA polymerase. Four non-structural polypeptides have been identified (NCVP5, NCVP4, NCVP2, NCVP3); very little is known about their biological role.
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PMID:[Rotaviruses: structure and function of the principal polypeptides]. 255 46

The simian rotavirus SA11 genome segment 10 codes for a nonstructural glycoprotein, NS28, that has been hypothesized to be involved in budding of viral particles into the endoplasmic reticulum (ER) membrane. Previous studies had suggested that NS28 is an integral membrane protein of the ER, possibly a transmembrane protein. We have examined the topography of NS28 inserted in microsomal membranes following cell-free translation of genome segment 10 transcripts. These transcripts were obtained either by hybrid selection of mRNA synthesized by the endogenous viral RNA polymerase or by in vitro transcription of genome segment 10 cDNA using SP6 polymerase. Full-length and truncated gene 10 transcripts were translated in a cell-free system supplemented with dog pancreatic microsomes. The existence of a cytoplasmic domain of the translation product was demonstrated by protease protection experiments. An 18,000 (18K) mol wt glycosylated polypeptide was protected from digestion with proteinase K and trypsin, whereas chymotrypsin digestion yielded a 23K mol wt glycosylated polypeptide. Correlation of these biochemical data with the known sequence of NS28 suggests that a 10K mol wt hydrophilic, carboxy-terminal fragment (from amino acid number 86 to amino acid number 175) of this glycoprotein is exposed on the cytoplasmic side of the ER membrane. A model of how NS28 folds in the ER membrane is proposed.
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PMID:Topography of the simian rotavirus nonstructural glycoprotein (NS28) in the endoplasmic reticulum membrane. 283 61

The M2 protein of influenza A virus is a small integral membrane protein of 97 residues that is expressed on the surface of virus-infected cells. M2 has an unusual structure as it lacks a cleavable signal sequence yet contains an ectoplasmic amino-terminal domain of 23 residues, a 19 residue hydrophobic transmembrane spanning segment, and a cytoplasmic carboxyl-terminal domain of 55 residues. Oligonucleotide-mediated deletion mutagenesis was used to construct a series of M2 mutants lacking portions of the hydrophobic segment. Membrane integration of the M2 protein was examined by in vitro translation of synthetic mRNA transcripts prepared using bacteriophage T7 RNA polymerase. After membrane integration, M2 was resistant to alkaline extraction and was converted to an Mr approximately equal to 7,000 membrane-protected fragment after digestion with trypsin. In vitro integration of M2 requires the cotranslational presence of the signal recognition particle. Deletion of as few as two residues from the hydrophobic segment of M2 markedly decreases the efficiency of membrane integration, whereas deletion of six residues completely eliminates integration. M2 proteins containing deletions that eliminate stable membrane anchoring are apparently not recognized by signal recognition particles, as these polypeptides remain sensitive to protease digestion, indicating that in addition they do not have a functional signal sequence. These data thus indicate that the signal sequence that initiates membrane integration of M2 resides within the transmembrane spanning segment of the polypeptide.
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PMID:Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus. 283 32

The primary structure of the messenger RNA coding for cytosolic phosphoenolpyruvate carboxykinase was determined by sequencing cDNA and genomic DNA and by primer extension of the mRNA. The molecule is 2624 nucleotides in length; this includes 143 nontranslated nucleotides at the 5' end and 615 nontranslated nucleotides at the 3' end. The 3' nontranslated sequence contains a 102-base pair region of alternating purine-pyrimidine nucleotides (the majority of which are UpG dinucleotides), several direct repeats and palindromic sequences, and 8 CpG dinucleotides. The corresponding segment of the phosphoenolpyruvate carboxykinase gene thus has characteristics which favor the formation of Z-DNA. The amino acid sequence of phosphoenolpyruvate carboxykinase was deduced from the mRNA sequence and confirmed by fast atom bombardment mass spectrometric analysis of peptides generated with trypsin and Staphylococcus aureus V8 protease. The protein consists of 621 amino acids and has a molecular weight of 69,289. Charon 4A lambda bacteriophage clones containing genomic DNA coding for phosphoenolpyruvate carboxykinase were isolated from a library of partial HaeIII digests of rat liver DNA. Two clones, lambda PC112 and lambda PC103, contained the entire coding region in 15-kilobase inserts and were used to subclone the gene into pBR322 as EcoRI, BamHI, or SstI-KpnI fragments. Using these subclones, the structure of the phosphoenolpyruvate carboxykinase gene was determined by S1 nuclease mapping, R-loop analysis, and DNA sequencing. The gene is composed of 10 exons and 9 introns with a total length of 6.0 kilobases. The transcription initiation site of the gene was determined by a combination of in vitro transcription in a HeLa cell lysate system, primer extension of mRNAPEPCK, and S1 nuclease mapping. In vitro transcription of purified DNA templates revealed three RNA polymerase II-dependent start sites. Two sites were separated by 600 base pairs on the coding strand and the third site was on the noncoding strand. The products of S1 nuclease mapping and primer extension from a BglII site were compared in order to determine which of the coding strand initiation sites was expressed in vivo. In both cases a 69-base pair fragment was generated and the 5' end of this corresponded to a thymidine residue identified in a sequence ladder of the genomic DNA coding strand. We conclude that mRNAPEPCK synthesis initiates with an adenine residue 69 base pairs 5' of the BglII site; this corresponds to the 3' most transcription initiation site determined in vitro.
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PMID:Rat hepatic cytosolic phosphoenolpyruvate carboxykinase (GTP). Structures of the protein, messenger RNA, and gene. 299 87


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