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)

Nucleophosmin/B23 is a nucleolar phosphoprotein which forms oligomers. To determine the domain essential for oligomer formation, various deletion and point mutation clones of nucleophosmin/B23 were constructed. Nucleophosmin/B23 and the mutant proteins were produced by (a) coupled in vitro transcription and translation and (b) expression in Escherichia coli with T7 RNA polymerase expression vector (pET-8c). Nucleophosmin/B23 synthesized in vitro has the same peptide map as that synthesized in HeLa cells. Similarly, it formed oligomers which could be detected in SDS/PAGE and were cross-linked with nitrogen mustard in vivo. Substitution of Met5, Met7, and Met9 with Leu or deletion of five amino acids at the C-terminus abolished the oligomerization. Deletion of portions of amino acids in the middle of the molecule (amino acid residues 83-152, 117-186 and 185-240) had little effect on the oligomerization. Co-expression of the N- and C-terminal mutant clones in vitro did not produce oligomers. These results indicate that intra-molecular interactions with both the N- and C-terminal domains are essential for oligomer formation.
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PMID:Formation of nucleophosmin/B23 oligomers requires both the amino- and the carboxyl-terminal domains of the protein. 191 43

We previously described the purification and characterization of E1BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E1BF was selectively phosphorylated. The labeled phosphate could be removed from the E1BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase. Elution of the protein from the E1BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with [gamma-32P] ATP resulted in the selective labeling of the 79-kDa band. The E1BF-associated protein kinase did not phosphorylate casein or histone H1. Fraction DE-B, a preparation containing RNA polymerase I and all polymerase I transcription factors (including E1BF), lost polymerase I transcriptional activity when treated with phosphatase. The phosphatase-induced inactivation of polymerase I activity associated with fraction DE-B could be reversed by the addition of purified E1BF. Treatment of purified E1BF with heat, SDS, or an ATP affinity analog eliminated its capacity to reactivate dephosphorylated fraction DE-B. These data demonstrate that (i) polymerase I promoter-binding factor E1BF contains an intrinsic substrate-specific protein kinase and (ii) E1BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation. Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.
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PMID:E1BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription. 192 88

RNase E is a major endonucleolytic RNA processing enzyme in Escherichia coli. We have sequenced a 3.2 kb EcoRI-BamHI fragment encoding the rne gene, and identified its reading frame. Upstream from the gene, there are appropriate consensus sequences for a putative promoter and a ribosome binding site. We have translated this gene using a T7 RNA polymerase/promoter system. We determined 25 amino acids from the N-terminal of the translated product and they are in full agreement with the DNA sequence. The translated product of the rne gene migrates in SDS containing polyacrylamide gels as a 110,000 Da polypeptide, but the open reading frame found in the sequenced DNA indicates a much smaller protein. The entity that migrates as a 110,000 Da contains RNA, which could account, at least partially, for the migration of the rne gene product in SDS containing polyacrylamide gels.
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PMID:Sequencing and expression of the rne gene of Escherichia coli. 201 93

Phosphorothioate-containing RNAs were generated by transcription of coliphage T7 DNA using the Sp diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates) and T7 RNA polymerase. RNAs in which a single nucleotide was substituted by the corresponding nucleoside phosphorothioate functioned as mRNA in the cell-free translation systems prepared from Escherichia coli and from an extreme thermophilic bacterium, Thermus thermophilus. This substitution increased the efficiency of protein synthesis by stabilizing the mRNAs in these systems. As the proportion of substituted nucleotides was increased, their mRNA activity was decreased accordingly. As judged from the analysis by SDS-polyacrylamide gel-electrophoresis, the proteins synthesized using phosphorothioate-containing mRNAs as template were identical to those obtained with unsubstituted mRNAs. However, larger proteins which were barely detectable when unsubstituted mRNA was used were well represented when phosphorothioate-RNA was used instead. The advantages in using the phosphorothioate-mRNAs in the in vitro translation systems are discussed.
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PMID:Phosphorothioate-containing RNAs show mRNA activity in the prokaryotic translation systems in vitro. 201 26

The structure of the rat homologue of the RNA polymerase I transcription factor UBF was investigated. The sequence of the protein was deduced from the sequence of overlapping cDNAs isolated from a cDNA library and from clones of the products generated by the polymerase chain reaction from random-primed, first-strand cDNA. The sequences of these clones indicated that there were two mRNAs for UBF and that the encoded proteins were similar but not identical. One form of rat UBF was essentially identical to human UBF. The second class of UBF mRNA contained an in-frame "deletion" in the coding region that results in the deletion of 37 amino acids from the predicted protein sequence. This deletion reduces the predicted molecular size of the encoded form of UBF by approximately 4400 from 89.4 kDa to 85 kDa and significantly alters the structure of one of the four HMG-1 homology regions (HMG box-2) in that form of UBF. Evidence for the existence of two mRNAs in rat cells was confirmed by a probe protection assay, and we provide evidence that other vertebrate cells contain these same two forms of UBF mRNA. These results are consistent with the observation that UBF purified from four different vertebrates migrates as two bands upon SDS/PAGE. It has been hypothesized that the HMG motifs are the DNA-binding domains of UBF. Altering one of these "boxes," as in the second form of UBF, may alter the functional characteristics of the transcription factor. Thus, the existence of different forms of UBF may have important ramifications for transcription by RNA polymerase I.
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PMID:Identification of two forms of the RNA polymerase I transcription factor UBF. 201 38

Plasmid pBR322 and its derivative containing strong promoter phi 10 of bacteriophage T7 RNA polymerase were used as vectors. A fragment of bacteriophage T7 DNA which was digested with two restriction endonucleases (AvaII and HaeIII) was cloned in the BamHI site of plasmid pBR322 and its derivative pAR951, respectively. The inserted DNA is a segment of 632 base pairs containing the complete coding sequence of both T7 gene 3.5 and weak promoter phi 3.8 for bacteriophage T7 RNA polymerase. The function of T7 gene 3.5 is known to code for bacteriophage T7 lysozyme. Transformants that carry the recombinant plasmid were tested for intracellular lysozyme by adding CHCl3. Both cloned strains produce active T7 lysozyme. The gene product, T7 3.5 protein, was analyzed by 10 -20% gradient polyacrylamide- SDS electrophoresis. The result showed that the expression of inserted T7 gene 3.5 in pBR322 derivative is stronger than that in pBR322.
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PMID:Cloning of the bacteriophage T7 lysozyme gene. 210 4

The Semliki-Forest-virus-specific nonstructural proteins are translated as a large polyprotein (2431 amino acid residues), from which the mature polymerase components nsP1, nsP2, and nsP4 are released by proteolytic cleavages. The complete ns polyprotein (P1234) can be cleaved in two alternative ways yielding either P123 (with sequences of nsP1, nsP2 and nsP3) and nsP4 or P12 (nsP1 plus nsP2) and P34 (nsP3 plus nsP4). We studied the possible autoproteolytic role of nsP4 involved in the cleavage between nsP3 and nsP4 in an in vitro transcription-translation system. cDNAs encoding P34 precursor and shorter precursor protein segments covering the nsP3-nsP4 cleavage region, were cloned under the T7 RNA polymerase promoter. The mRNAs synthesized in vitro were capped and translated in rabbit reticulocyte lysates. The translational products were analyzed by SDS/PAGE. The precursor proteins containing nsP4 sequences were cleaved yielding the products with expected sizes, indicating that the cleavage took place at the nsP3-nsP4 junction. By deleting and truncating the cDNA coding for nsP4, the proteolytic activity was mapped within the 102 amino-terminal amino acids of nsP4. The cleavage between nsP3 and nsP4 can be inhibited by pepstatin A and probably takes place in cis, since exogenously added nsP4 was unable to mediate it.
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PMID:The Semliki-Forest-virus-specific nonstructural protein nsP4 is an autoproteinase. 213 9

Bacteriophage T7 expresses a serine/threonine-specific, cAMP-independent protein kinase activity encoded by the early gene 0.7. The phosphoproteins specifically resulting from gp0.7 protein kinase expression in T7-infected Escherichia coli have been examined by one-dimensional, SDS-polyacrylamide gel electrophoresis. Only seven major, stable phosphoproteins dependent on gp0.7 protein kinase expression are observed. Two of the gp0.7 protein kinase-specific phosphoproteins observed have been previously identified: the beta' subunit of RNA polymerase and the RNA processing enzyme RNase III. The gp0.7-catalyzed protein phosphorylation activity appears at 9-11 min postinfection at 30 degrees. The new phosphoproteins have a metabolic stability comparable to that of uninfected cell phosphoproteins. T7 protein kinase expression causes the phosphorylation of the same, limited set of proteins in B, C, or K strains of E. coli. Expression of the T3 and BA14 phage protein kinase activities also produces the same phosphoproteins.
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PMID:Protein kinase of bacteriophage T7 induces the phosphorylation of only a small number of proteins in the infected cell. 218 69

The gene of catalytic domain of the protein kinase of RSV-scr was cloned into the BamHI cloning site of translation vector pET-8c which containing T7 RNA polymerase promotor, and transformed BL21 (DE3) pLys S (Studier and Moffatt, 1986). The putative molecular weight of the protein was about 33 kd as evaluated on the basis of its nucleotide size showed the identical mobility in SDS-polyacrylamide gel electrophoresis. However, yield of protein production was not high, probably, because of its instability in Escherichia coli.
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PMID:Subcloning and trial of expression of the protein kinase catalytic domain of RSV-src gene. 221 79

Various agents were tested for their effects on microbial proteases, which activity was monitored by the analysis of cleaved peptide bands in SDS-polyacrylamide gel electrophoresis. Using casein as a substrate, fungal protease (type XIX) was inhibited by the phenyl methyl sulphonyl fluoride, chymostatin, antipain and leupeptin, while bacterial protease (type XXVI) was inhibited by phosphatidyl glycerol, phosphatidyl inositol and sphingosine. MS2 RNA exerted minor inhibition on the bacterial proteolysis of regulatory subunits of cyclic AMP-dependent protein kinase (A-PK). The cleavage of DNA binding protein by both proteases was inhibited, in the presence of MS2 RNA and lambda DNA. In comparison, phosphatidyl serine slightly stimulated the fungal protease on the cleavage of ribonuclease T1. RNA polymerase is a good substrate of the bacterial protease as indicated by the generation of multiple cleaved peptide fragments, whereas alkaline phosphatase is not susceptible to proteolysis.
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PMID:A further study on the regulation of microbial proteases. 222 36


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