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)

Conveniently situated PstI sites were used to delete a major segment from the C-peptide coding region of a human pre-pro-insulin cDNA. The resultant mutant cDNA encoded a protein with the structure: pre-peptide B chain--Arg-Arg-Glu-Ala-Glu-Asp-Leu-Gln-Lys-Arg-A chain. Normal and mutant human pre-pro-insulin cDNAs were used as templates for the synthesis of mRNA in a reaction catalysed by T7 RNA polymerase. The mRNAs were then microinjected into Xenopus oocytes to determine the effect of the deletion on the secretion of pro-insulin. When normal pre-pro-insulin mRNA was microinjected, pre-pro-insulin was processed to pro-insulin, which in turn was secreted into the media. When the mutant pre-pro-insulin mRNA was microinjected, however, mutant pro-insulin could be detected in the oocytes but at a much lower level than the normal pro-insulin. No mutant pro-insulin could be detected in the media. The stability of the mRNAs in the oocytes was investigated by microinjecting [32P]mRNA. 24 and 48 h after microinjection, the recovery of [33P]mRNA from the oocytes was 95 and 24% and 20 and 16% of that injected, for the normal and mutant mRNAs, respectively. In a cell-free translation system supplemented with dog pancreatic microsomal membranes, the pre-peptide was cleaved from the normal pre-pro-insulin but not from the mutant pre-pro-insulin. These results suggest that C-peptide plays an important role in the segregation of pro-insulin within and transport through the cellular secretory pathway.
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PMID:Expression of normal and mutant human pre-pro-insulins in Xenopus oocytes. 284 Sep 76

We used a synthetic double-stranded oligonucleotide to introduce amino acid substitutions into the proteinase 3C region of a poliovirus type 1 cDNA clone. The six different mutant viruses recovered exhibited a small-plaque phenotype when assayed on HeLa cells. Further investigation revealed that all the mutations (with the exception of one) yielded P3 region proteins that displayed altered mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A conservative Val----Ala change at amino acid 54 of the proteinase resulted in a virus that was deficient in the production of the mature viral RNA polymerase 3D. Although this mutant achieved less than one-half of the wild-type levels of RNA synthesis during the course of infection, it still grew to nearly wild-type titers.
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PMID:Site-directed mutagenesis of proteinase 3C results in a poliovirus deficient in synthesis of viral RNA polymerase. 303 16

We have used an in vitro transcription-translation system to study initial protein processing events of the rat substance P/neurokinin A gene products. cDNA clones for three different mRNA species, which are derived by differential RNA splicing, were subcloned into a plasmid, pGEM1, which contains the promoter for the bacteriophage SP6 RNA polymerase. In vitro synthesized mRNAs for alpha-, beta-, and gamma-preprotachykinin were translated in a wheat germ or rabbit reticulocyte cell-free system. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the translated protein products migrate consistent with the deduced molecular masses of alpha (13,035 Da)-, beta (15,003 Da)-, and gamma (13,343 Da)-preprotachykinin. The addition of dog pancreatic microsomal membranes to either cell-free translation system causes the production of a protease-resistant form of each of the three preprotachykinins which migrates with an apparent increase in molecular mass of approximately 2,000 Da. Each of these modified preprotachykinins lacks the putative signal peptide of the prepro- form, with signal peptidase cleavage occurring after the alanine residue at position 19. Both the prepro- and proforms of each tachykinin precursor molecule are recognized by antiserum R-140, an antiserum specific for the mid-portion of the undecapeptide substance P. The most likely explanation for the apparent increase in molecular mass is anomalous electrophoretic migration, since beta-preprotachykinin mRNA lacking the signal peptide encoding sequence is translated, in the absence of microsomal membranes, into a protein with the same apparent molecular mass as the modified form of beta-preprotachykinin. Therefore, each of the three preprotachykinin mRNAs are translatable, and their products are targeted to the secretory pathway by the presence of a cleavable signal peptide.
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PMID:Posttranslational processing of alpha-, beta-, and gamma-preprotachykinins. Cell-free translation and early posttranslational processing events. 304 2

The DNA sequence of the Bacillus subtilis DLG endo-beta-1,4-glucanase gene was determined, and the in vivo site of transcription initiation was located. Immediately upstream from the transcription start site were sequences closely resembling those recognized by B. subtilis sigma 43-RNA polymerase. Two possible ribosome-binding sites were observed downstream from the transcription start site. These were followed by a long open reading frame capable of encoding a protein of ca. 55,000 daltons. A signal sequence, typical of those present in gram-positive organisms, was observed at the amino terminus of the open reading frame. Purification of the mature exocellular beta-1,4-glucanase and subsequent amino-terminal protein sequencing defined the site of signal sequence processing to be between two alanine residues following the hydrophobic portion of the signal sequence. The probability of additional carboxy-terminal processing of the beta-1,4-glucanase precursor is discussed. S1 nuclease protection studies showed that the amount of beta-1,4-glucanase mRNA in cells increased significantly as the culture entered the stationary phase. In addition, glucose was found to dramatically stimulate the amount of beta-1,4-glucanase mRNA in vivo. Finally, the specific activities of purified B. subtilis DLG endo-beta-1,4-glucanase and Trichoderma reesei QM9414 endo-beta-1,4-glucanase (EC 3.2.1.4) were compared by using the noncrystalline cellulosic substrate trinitrophenyl-carboxymethyl cellulose.
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PMID:Endo-beta-1,4-glucanase gene of Bacillus subtilis DLG. 310 28

Recently, we reported the molecular cloning and nucleotide sequence analysis of a gene from Rickettsia rickettsii that codes for a 17-kilodalton antigen (17K antigen) and is preceded by sequences closely resembling the -10 and -35 consensus sequences for recognition by Escherichia coli RNA polymerase (Anderson et al., J. Bacteriol. 169:2385-2390, 1987). Experiments described in this report indicate that the start sites for initiating transcription of the 17K antigen gene are identical in the E. coli clone and in intact R. rickettsii. In each case, initiation was shown to begin 9 bases downstream of the presumed Pribnow box sequence (TATACT). A 169-base-pair fragment containing the promoter sequence initiated transcription in both directions when cloned into an E. coli promoter probe vector. The rickettsial fragment was found to contain sequences identical to the -10 region (but not the -35 region) of the E. coli promoter consensus sequence directed away from the 17K antigen gene. The amino-terminal portion (residues 17 to 20) of the deduced amino acid sequence for the 17K antigen contained the tetrapeptide Leu-Gln-Ala-Cys, a sequence that conforms favorably to those described for lipid modification and cleavage by lipoprotein signal peptidase II. The 17K antigen produced by the E. coli clone was shown to be labeled with [3H]palmitate and [3H]glycerol, indicative of lipid modification. In vitro mutagenesis designed to alter the cysteine at residue 20 to a glycine abolished incorporation of [3H]palmitate, suggesting that posttranslational modification occurs via a mechanism similar to that described for other gram-negative bacterial lipoproteins.
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PMID:Expression of the gene encoding the 17-kilodalton antigen from Rickettsia rickettsii: transcription and posttranslational modification. 313 29

One or more of the five acidic amino-terminal residues of skeletal muscle actin have been implicated as being important in a number of actin-related processes. We have constructed a series of actins containing mutations at Asp3 and Asp11 and tested these mutant proteins for their ability to bind to DNase I-agarose, polymerize with rabbit skeletal muscle actin, undergo amino-terminal processing, and bind to the myosin-S1 subfragment. The mutant actins were expressed in vitro using a coupled transcription/translation system which involves the synthesis of mutant RNAs with SP6 RNA polymerase followed by their translation in a rabbit reticulocyte lysate. When Asp3 was changed to Ala, His, or Asn there was no difference in the tested properties as compared to wild type actin. These results suggest that an acidic residue at position 3 is not critical for the actin functions measured. When Asp11 was changed to Glu, Asn, or His or if the conserved Asp-Asn sequence at positions 11 and 12 was reversed, the mutants were able to copolymerize with rabbit skeletal muscle actin and be cross-linked to myosin-S1 to nearly the same extent as wild type actin. However, the amount of in vitro-synthesized actin capable of binding to DNase I-agarose with high affinity or undergoing amino-terminal processing was reduced significantly relative to the wild type actin synthesized in vitro. The Asp11 mutants ran anomalously on native polyacrylamide gels suggestive of a conformational change induced in the actin. Together, these results suggest that Asp11 may be important in proper actin folding and function.
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PMID:Studies on the role of actin's aspartic acid 3 and aspartic acid 11 using oligodeoxynucleotide-directed site-specific mutagenesis. 319 44

Chimeric phage-plasmid expression vectors were constructed from pUC18/19 plasmids by cloning a single-stranded DNA (ssDNA) origin of replication from bacteriophage f1 and inserting a bacteriophage T7 promoter within the beta-galactosidase gene. A T7 promoter permits in vivo or in vitro expression of single proteins by the translation of T7 RNA polymerase transcripts. Insertional inactivation of the T7 promoter-containing beta-galactosidase gene permits a simple blue-to-white color cloning assay. Compared with several helper phages that were examined, superinfection with M13K07 resulted in the highest yields of the pTZ plasmids as ssDNA viral particles. These ssDNA promoter plasmids are uniquely suited for protein engineering because they simplify cloning, oligonucleotide directed mutagenesis, verification by enzymatic sequence analysis, and expression of mutant proteins from a single vector. These vectors were utilized to eliminate an efficient transcriptional terminator of T7 RNA polymerase in the cDNA of bovine preproparathyroid hormone by oligonucleotide directed mutagenesis. The mutation changed the codon for phenylalanine-19 in the signal peptide to alanine. In a cell-free system the mutant cDNA transcripts were translated into preproparathyroid hormone, which was converted to proparathyroid hormone in the presence of microsomal membranes.
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PMID:Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. 350 89

The effect of substitution of L and D amino acids in amatoxin analogues is discussed in this paper. The structure of the analog where D-alanine substitutes for glycine in position 7 has been worked out in solution by two-dimensional NMR methods using a 500 MHz instrument. The combined use of COSY and NOESY two-dimensional spectra allows a clear assignment of the resonances. The use of the coupling constants permits the calculation of the phi angles of the backbone. The NOE effects reveal the through-space contacts between protons of different peptide units, thus determining the rigidity of the amatoxin structure. On these grounds it has been possible to elucidate the conformation of the amatoxin analogue that resembles very closely that of beta-amanitin, thus explaining the high inhibitory activity toward RNA polymerase B.
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PMID:Synthetic amatoxin analogue. A two-dimensional proton NMR study of S-deoxy-(Ile3)-(D-Ala7)-amaninamide. 369 61

Short interspersed repetitive DNA sequences (SINEs) are the major component of dispersed repetitive DNA in all mammalian genomes. Most SINEs contain an intragenic RNA polymerase III promoter that initiates transcription at the 5' end of the repeated DNA sequence and which has been proposed to facilitate the transposition and amplification of these sequences by an RNA-intermediate mechanism. We have discovered several SINE families in the prosimian Galago crassicaudatus which have promoter regions similar to transfer RNA genes. To determine the relationship between Galago SINEs and mammalian tRNA genes, we have compared their sequences. Here, we demonstrate that the Galago monomer and type II SINE families are 68 and 62% homologous, respectively, with a human methionine tRNA gene. We have extended our analysis to include the rat identifier and mouse B2 families and show that their sequences are closely related to alanine and serine tRNA genes, respectively. Our observations suggest that many mammalian SINE families are amplified tRNA pseudogenes.
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PMID:Repeat sequence families derived from mammalian tRNA genes. 385 Nov 63

The nucleotide sequence of a 2.14 kb fragment of Bacillus subtilis DNA containing the citG gene encoding fumarase was determined using the dideoxy chain termination method. The citG coding region of 1392 base pairs (464 codons) was identified, and the deduced Mr (50425) is in good agreement with that of the protein identified from expression in Escherichia coli maxicells. There is no sequence homology between the B. subtilis and E. coli fumarases. Overlapping potential promoter sequences have been identified for sigma 28, sigma 37 and sigma 55 RNA polymerase holoenzymes. The DNA fragment also contains the proximal part of the gerA locus, responsible for L-alanine-sensitive spore germination.
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PMID:Complete nucleotide sequence of the fumarase gene (citG) of Bacillus subtilis 168. 392 30


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