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)

The structural gene for the Bacillus stearothermophilus glycogen branching enzyme (glgB) was cloned in Escherichia coli. Nucleotide sequence analysis revealed a 1917 nucleotide open reading frame (ORF) encoding a protein with an Mr of 74787 showing extensive similarity to other bacterial branching enzymes, but with a shorter N-terminal region. A second ORF of 951 nucleotides encoding a 36971 Da protein started upstream of the glgB gene. The N-terminus of the ORF2 gene product had similarity to the Alcaligenes eutrophus czcD gene, which is involved in cobalt-zinc-cadmium resistance. The B. stearothermophilus glgB gene was preceded by a sequence with extensive similarity to promoters recognized by Bacillus subtilis RNA polymerase containing sigma factor H (E - sigma H). The glgB promoter was utilized in B. subtilis exclusively in the stationary phase, and only transcribed at low levels in B. subtilis spoOH, indicating that sigma factor H was essential for the expression of the glgB gene in B. subtilis. In an expression vector, the B. stearothermophilus glgB gene directed the synthesis of a thermostable branching enzyme in E. coli as well as in B. subtilis, with optimal branching activity at 53 degrees C.
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PMID:Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (glgB) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis. 174 26

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

Yeast nuclear RNA polymerases are multisubunit enzymes that contain in common some small subunits. We show that the smallest, a 10-kDa component of three enzymes (A10, B10, and C10), is heterogeneous. In each case, it can be resolved into two distinct polypeptides (alpha and beta) by reverse-phase chromatography. A10 alpha, B10 alpha, and C10 alpha were indistinguishable on the basis of their electrophoretic and chromatographic behaviors, characteristic silver staining, and tryptic peptide analysis. All three polypeptides are blocked at their amino termini. By the same criteria, A10 beta, B10 beta, and C10 beta were also indistinguishable. The amino-terminal sequence of A10 beta and C10 beta corresponded to that of subunit B10 recently cloned by Woychik and Young (Woychik, N. A., and Young, R. A. (1990) J. Biol. Chem. 265, 17816-17819). Thus, the three forms of RNA polymerase share two additional and distinct polypeptides, ABC10 alpha and ABC10 beta, that therefore can be considered bona fide subunits of these enzymes. Interestingly, these two subunits bind zinc.
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PMID:Two additional common subunits, ABC10 alpha and ABC10 beta, are shared by yeast RNA polymerases. 174 81

Rat liver protein disulfide isomerase (PDI) catalyzes the oxidative folding of proteins containing disulfide bonds. We have developed an efficient method for its overproduction in Escherichia coli. Using a T7 RNA polymerase expression system, isolated yields of 15-30 mg/liter of recombinant rat PDI are readily obtained. Convenient purification of the enzyme from E. coli lysates involves ion-exchange (DEAE) chromatography combined with zinc chelate chromatography. The recombinant PDI shows catalytic activity identical to that of PDI isolated from bovine liver in both the reduction of insulin and the oxidative folding of ribonuclease A. The enzyme is expressed in E. coli as a soluble, cytoplasmic protein. After complete reduction and denaturation in 6 M guanidinium hydrochloride, PDI regains complete activity within 3 min after removal of the denaturant, implying that disulfide bonds are not essential for the maintenance of PDI tertiary structure. Both the protein isolated from E. coli and the protein isolated from liver contained free cysteine residues (1.8 +/- 0.2 and 1.4 +/- 0.3 SH/monomer, respectively).
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PMID:Expression and purification of recombinant rat protein disulfide isomerase from Escherichia coli. 182 89

Bacteriophage Mu controls an unusual DNA-modification function encoded by the mom gene, which is located in an operon that consists of two overlapping genes. The com gene, located proximal to the 5' end of the common mRNA transcript, encodes a polypeptide of 62 amino acids that is required for translation of mom. Analysis of the derived amino acid sequence reveals that Com contains zinc-binding finger motifs, suggesting that Com may be a zinc-activated regulatory protein. Atomic absorption analysis showed that there is about one zinc bound per molecule of Com. We have subcloned the com gene into an expression vector and thus have overproduced and purified the Com protein. By gel retardation analysis with various 32P-labeled RNAs (made by in vitro transcription with T7 RNA polymerase), we show that Com binds specifically to com-mom mRNA. A single C----U substitution mutation, located 26 nucleotides upstream from the mom translation start codon, abolishes Com binding. The nature of the Com target sequence was deduced from in vitro footprinting analyses. The results are consistent with the existence of a complex stem-loop structure within the overlap of the com-mom open-reading-frames. Com binding to its target site results in the destabilization of a proposed translation-inhibitor stem-loop (TIS) to expose the Shine-Dalgarno sequence and mom translation initiation codon. This suggests that Com interaction with a specific site on its cognate mRNA alters the mRNA secondary structure to activate translation of mom.
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PMID:Com, the phage Mu mom translational activator, is a zinc-binding protein that binds specifically to its cognate mRNA. 183 88

The rapid induction of thionein (apometallothionein) by many endogenous stimuli such as steroid hormones, cytokines, and second messengers suggests that this cysteine-rich, metal binding protein participates in an as yet undefined role in cellular regulatory processes. This study demonstrates with DNA and RNA binding assays and in vitro transcription measurements that thionein suppresses the binding of the Xenopus laevis zinc finger transcription factor IIIA (TFIIIA) to 5S RNA and to the 5S RNA gene and abrogates the capacity of TFIIIA to initiate the RNA polymerase III-catalyzed synthesis of 5S RNA. The effect is reversed by the addition of zinc and is not observed in the TFIIIA-independent transcription of a tRNA gene by the same RNA polymerase. In view of the strong tendency of thionein to complex posttransition metals such as zinc, one effect of its enhanced synthesis in vivo could be to reduce the intracellular disposability of zinc and thus modulate the actions of zinc-dependent enzymes and proteins, most notably those of the zinc finger transcription factors.
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PMID:Zinc transfer from transcription factor IIIA fingers to thionein clusters. 183 92

Starting with two temperature-sensitive mutants (rpa190-1 and rpa190-5) of Saccharomyces cerevisiae, both of which are amino acid substitutions in the putative zinc-binding domain of the largest subunit (A190) of RNA polymerase I, we have isolated many independent pseudorevertants carrying extragenic suppressors (SRP) of rpa190 mutations. All the SRP mutations were dominant over the corresponding wild-type genes. They were classified into at least seven different loci by crossing each suppressed mutant with all of the other suppressed mutants and analyzing segregants. SRP mutations representing each of the seven loci were studied for their effects on other known rpa190 mutations. All of the SRP mutations were able to suppress both rpa190-1 and rpa190-5. In addition, one particular suppressor, SRP5, was found to suppress two other rpa190 mutations as well as an rpa190 deletion. Southern blot analysis combined with genetic crosses demonstrated that SRP5 maps to a region on chromosome XV loosely linked to rpa190 and represents a transposed mutant gene in two copies. Analysis of the A190 subunit by using anti-A190 antiserum indicated that the cellular concentration of A190 and hence of RNA polymerase I decreases in rpa190-1 mutants after a shift to 37 degrees C and that in the mutant strain carrying SRP5 this decrease is partially alleviated, presumably because of increased synthesis caused by increased gene dosage. These results suggest that the zinc-binding domain plays an important role in protein-protein interaction essential for the assembly and/or stability of the enzyme, regardless of whether it also participates directly in the interaction of the assembled enzyme with DNA.
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PMID:Suppressor analysis of temperature-sensitive RNA polymerase I mutations in Saccharomyces cerevisiae: suppression of mutations in a zinc-binding motif by transposed mutant genes. 184 71

We have demonstrated earlier that human cells contain nuclear protein interacting with conserved GC-rich sequence motifs of human Alu-family DNA repeats. One of these sequences is located in the region between elements A and B of bipartite RNA polymerase III promoter of Alu (AB-region). In this study we have used a DNase I footprinting assay with an Alu restriction subfragment covering AB-region, as well as a gel mobility shift assay with appropriate synthetic oligonucleotides to analyse in more detail the interaction of the protein with AB-region. We have also used antibodies raised against a zinc-finger peptide to examine the presence of a zinc-finger in the Alu-binding protein. The results indicate that AGG triplets may be important for high-affinity binding of the protein to DNA, and that the Alu-binding protein is a zinc-finger protein.
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PMID:Binding specificity of human nuclear protein interacting with the Alu-family DNA repeats. 185 21

Two open reading frames on a 3.7-kb BglII-XbaI fragment which encodes the Staphylococcus aureus cadA cadmium (and zinc) resistance determinant of plasmid pI258 were identified (G. Nucifora, L. Chu, T. K. Misra, and S. Silver, Proc. Natl. Acad. Sci. USA 86:3544-3548, 1989). The [35S]methionine-labelled protein products of the 727-amino-acid CadA ATPase and of the 122-amino-acid CadC polypeptide in Escherichia coli were identified by using the T7 RNA polymerase-promoter expression system. A truncated CadA polypeptide (402 amino acids) did not confer resistance in S. aureus but was expressed in E. coli under control of the T7 RNA polymerase-promoter. Removal of 678 nucleotides from the 5' end of the published sequence (which includes the cadA promoter) abolished resistance to cadmium, whereas a 146-nucleotide-shorter deletion was without effect. The cadC gene is needed in addition to cadA for full resistance to cadmium in S. aureus and Bacillus subtilis. cadC functions both in cis and in trans.
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PMID:A second gene in the Staphylococcus aureus cadA cadmium resistance determinant of plasmid pI258. 193 59

The zinc-binding subunits of yeast RNA polymerase A(I) and B(II) have been identified by a zinc-blotting technique. The two largest subunits of each enzyme (A190, A135, B220, and B150), as well as A12.2, A10, B44.5, B12.6, and B10, bind 65Zn(II). Predicted zinc-binding motifs have been noted in the NH2-terminal part of B220 and the COOH-terminal region of B150 subunits. Subdomains encompassing these motifs have been overproduced as MalE-fusion proteins and shown to retain zinc binding activity. Site-directed mutagenesis in the predicted metal-binding domain of B150 demonstrated its role in zinc binding. Mutations of cysteine residues C1163, C1166, C1182, and C1185 affected 65Zn2+ binding in vitro and caused a lethal or thermosensitive phenotype for growth. The ability to bind zinc is not sufficient for function since mutations in vicinal residues not affecting zinc binding were either lethal or thermosensitive. The role of zinc in RNA polymerase structure and function is discussed in the light of the present results.
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PMID:Zinc-binding subunits of yeast RNA polymerases. 193 19


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