Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hammerhead ribozyme sequences were incorporated into a tyrosine tRNA (tRNA(Tyr)) and compared with nonembedded molecules. To increase the levels of ribozyme and control antisense in vivo, sequences were expressed from an autonomously replicating vector derived from African cassava mosaic geminivirus. In vitro, the nonembedded ribozyme cleaved more target RNA, encoding chloramphenicol acetyltransferase (CAT), than the tRNA(Tyr) ribozyme. In contrast, the tRNA(Tyr) ribozyme was considerably more effective in vivo than either the nonembedded ribozyme or antisense sequences, reducing CAT activity to < 20% of the control level. A target sequence (CM2), mutated to be noncleavable, showed no reduction in CAT activity in the presence of the tRNA(Tyr) ribozyme beyond that for the antisense construct. The reduction in full-length CAT mRNA and the presence of specific cleavage products demonstrated in vivo cleavage of the target mRNA by the tRNA(Tyr) ribozyme. The high titer of tRNA(Tyr) ribozyme was a result of transcription from the RNA polymerase III promoter and led to the high ribozyme/substrate ratio essential for ribozyme efficiency.
 ...
PMID:Effective ribozyme delivery in plant cells. 759 97

We have overproduced the leader peptidase from Escherichia coli in a high yield by using a T7 RNA polymerase/promoter system and purified the enzyme. This leader peptidase showed an apparent pH optimum of about 10 toward a synthetic peptide substrate, and was stable at temperatures below 40 degrees C. Kinetic studies indicated that one of the active site residues in the enzyme has a pKa value of approximately 7.5. The enzyme was rapidly inactivated by reaction with N-bromosuccinimide (NBS). When approximately two tryptophan residues were oxidized with NBS, the activity was almost completely lost and this inactivation was markedly prevented by a substrate. These NBS-reactive tryptophan residues were identified as Trp300 and Trp310 by a peptide mapping analysis. This indicates that Trp300 and/or Trp310 are critically important for the activity of the leader peptidase. On the other hand, the enzyme was scarcely inhibited by treatment with N-acetylimidazole, iodoacetic acid, 5,5'-dithiobis(2-nitrobenzoic acid), succinic anhydride, or 2,4,6-trinitrobenzenesulfonate. Diethylpyrocarbonate inhibited the enzyme; however, this inhibition did not seem to result from the modification of histidine residues. Thus, there seem to be no functionally important tyrosine, cysteine, or histidine residues or amino groups among the residues which readily react with these reagents. However, the enzyme was inactivated significantly by treatment with phenylglyoxal or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Therefore, some of the arginine residues and the carboxyl groups appear to be important for the enzyme activity.
 ...
PMID:Leader peptidase from Escherichia coli: overexpression, characterization, and inactivation by modification of tryptophan residues 300 and 310 with N-bromosuccinimide. 762 19

Our long-term goal is to define the catalytic domains of the L protein subunit of the Sendai virus RNA polymerase. An aberrant polyadenylation phenotype in the vesicular stomatitis virus tsG16 L protein mutant has recently been identified as a phenylalanine to serine change at amino acid 1488 (Hunt and Hutchinson, Virology 193, 786-793, 1993). To test if functional domains are conserved in the L proteins of negative-strand RNA viruses, we attempted to create a similar polyadenylation defect in the Sendai virus L protein. Nine different amino acid substitutions at the analogous site in the Sendai L protein (cysteine at amino acid 1571) were constructed by site-directed mutagenesis of the gene. Each mutant L protein was synthesized and bound to the Sendai P protein to form the P-L polymerase complex. While none of these L mutants exhibited a change in polyadenylation, the single amino acid changes yielded a variety of activities in vitro. Mutants containing valine, leucine, or phenylalanine at amino acid 1571, amino acids found naturally in the L proteins of other paramyxoviruses, yielded polymerases that had biological activity equal to or better than the wild-type (WT) polymerase. Serine or threonine substitutions in the L protein at this position also resulted in polymerases with nearly WT synthetic activity. In contrast, a glycine substitution significantly decreased overall polymerase activity, whereas a tyrosine substitution gave decreased transcription, but virtually no DI genome replication in vitro. The tyrosine-substituted polymerase may be unable to carry out the packaging step of replication, since DI leader RNA synthesis was normal in this mutant. Mutant L proteins with basic arginine or histidine substitutions were inactive in all viral RNA synthesis in vitro, although the polymerase complexes could bind the nucleocapsid template.
 ...
PMID:Alternative amino acids at a single site in the Sendai virus L protein produce multiple defects in RNA synthesis in vitro. 764 61

We have investigated the incorporation of 2'-deoxynucleoside-5'-O-(1-thiotriphosphates) into RNA transcripts using T7 RNA polymerase. With the exception of [alpha-S]dGTP, we obtained full-length transcripts of pre-tRNA(Phe) and pre-tRNA(Tyr) using an appropriate mixture of 2'-deoxynucleoside 5'-O-(1-thiotriphosphate) and the corresponding normal nucleoside triphosphate. The yields of the transcripts were comparable to those obtained with unmodified NTPs. Both substrates, [alpha-S]dTTP and [alpha-S]dATP, were inserted specifically. However, [alpha-S]dCTP was excluded at specific sites. We could not obtain transcripts using the deoxyguanosine derivative.
 ...
PMID:Enzymatic RNA synthesis with deoxynucleoside 5'-O-(1-thiotriphosphates). 767 87

The Escherichia coli DNA bending protein factor for inversion stimulation (FIS), is neither necessary nor responsible for the stimulation of transcription from the wild type promoter for the tyrT operon (encoding a species of tyrosine tRNA) that occurs upon resumption of exponential growth. This conclusion is unexpected given that the regulatory element required for optimal transcription of tyrT contains three binding sites for FIS protein. In addition, it is in apparent conflict with reports from other laboratories which have described FIS-dependent activation of the stable RNA promoters rrnB P1 and thrU(tufB) in vivo. However, tyrT transcription is stimulated in a FIS-dependent manner both in vivo and in vitro when promoter function is impaired by mutation of the promoter itself or by the addition of the polymerase effector guanosine 5'-diphosphate 3'-diphosphate. These conditions, which expose a requirement for activation of stable RNA synthesis by FIS, suggest that FIS serves an adaptive role permitting high levels of stable RNA transcription on nutritional shift-up when RNA polymerase levels are depleted. In principle such a mechanism could confer a significant selective advantage thus accounting for the conservation of FIS binding sites in the regulatory regions of stable RNA promoters.
 ...
PMID:The Escherichia coli FIS protein is not required for the activation of tyrT transcription on entry into exponential growth. 768 76

A series of plasmid vectors, pRSET A, B, and C, have been developed for high-level protein expression in prokaryotes and have been characterized. Based upon the T7 RNA polymerase-driven pET system, the pRSET vectors encode recombinant proteins as fusions with a multifunctional leader peptide containing a hexahistidyl sequence for purification on Ni(2+)-affinity resins, a tyrosine residue for radioiodination, and an enterokinase proteolytic cleavage site for leader peptide removal. Monoclonal antibodies (MAbs) to two epitopes on the leader peptide, which also contains amino acids 1-12 of the T7 gene 10 major capsid protein, were developed and provide for universal immunological detection of pRSET-expressed fusion proteins. Subcloning of protein-encoding DNA is facilitated by an 11-site polylinker which is offset for all three ribosomal reading frames, and an f1(+) origin of DNA replication permits single-stranded DNA synthesis for site-directed mutagenesis protocols. Representative fusion proteins overexpressed in Escherichia coli were successfully purified under both denaturing and nondenaturing conditions by single-step Ni2+ affinity chromatography. Purification was independent of recombinant protein solubility in sonicated or freeze-thawed E. coli lysates. Isolation of MAbs for selective recognition of either of two leader peptide epitopes was demonstrated by immunoprecipitation, but this selectivity was less evident under conditions for Western blotting. In combining the utility of T7 RNA polymerase-directed expression with several recent advances in protein purification and detection, the pRSET vectors will serve as a powerful resource for a variety of studies in protein biochemistry.
 ...
PMID:A multifunctional prokaryotic protein expression system: overproduction, affinity purification, and selective detection. 768 11

Ty3 is a retrotransposon of Saccharomyces cerevisiae that integrates just upstream of the transcription initiation site of genes transcribed by RNA polymerase III. Ty3 transcription is pheromone-inducible in haploid cells and is mating-type regulated in diploid cells. The specificity of Ty3 integration was exploited in the design of a novel target into which transposition of Ty3 elements could be selected. The target plasmid contains divergently oriented tRNA genes with 19 base pairs separating the two tRNA gene coding sequences. An inactive ochre suppressor tRNA(Tyr) gene with a modified transcription initiation region was used as the selectable marker and a tRNA(Val) (AAC) gene was used to direct Ty3 integration into the transcription initiation region of the suppressor tRNA(Tyr) gene. Integration of Ty3 activated expression of the suppressor tRNA gene, which resulted in suppression of ochre nonsense alleles ade2-101(0) and lys2-1(0) and allowed cell growth on selective medium. Based on the activity of this target, Ty3, under control of a galactose-inducible promoter and present on a high copy-number plasmid, was estimated to transpose into the genome at a rate of 5.6 x 10(-3) per cell division. We show here that induction of Ty3 transcription from its natural promoter results in transposition. Ty3 elements in strains of the a or alpha mating-type transposed efficiently to target plasmids in cells of the opposite mating-type. Thus, natural transposition of Ty3 is regulated temporally to occur in mating populations.
 ...
PMID:Ty3 transposes in mating populations of yeast: a novel transposition assay for Ty3. 770 53

In order to look more closely at a well-conserved region in T7 RNA polymerase (T7 RNAP) containing, as shown earlier, the functionally essential residues Pro-563 and Tyr-571, we used targeted mutagenesis to change those residues within this region that are invariant in all single-subunit RNA polymerases, and characterized the mutant enzymes in vitro. The most interesting finding of this study was the crucial importance of the acidic group of Asp-569. In addition, we have shown that the phenolic ring is the most significant functional group of Tyr-571, with the hydroxy group also contributing to promoter binding.
 ...
PMID:Targeted mutagenesis identifies Asp-569 as a catalytically critical residue in T7 RNA polymerase. 771 97

The RNA polymerase II carboxy-terminal domain (CTD) consists of tandem repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The CTD may participate in activated transcription through interaction with a high-molecular-weight mediator complex. Such a role would be consistent with observations that some genes are preferentially sensitive to CTD mutations. Here we investigate the function of the mouse RNA polymerase CTD in enhancer-driven transcription. Transcription by alpha-amanitin-resistant CTD-deletion mutants was tested by transient transfection of tissue culture cells in the presence of alpha-amanitin in order to inhibit endogenous RNA polymerase II. Removal of most of the CTD abolishes transcriptional activation by all enhancers tested, whereas transcription from promoters driven by Sp1, a factor that typically activates housekeeping genes from positions proximal to the initiation sites, is not affected. These findings show that the CTD is essential in mediating 'enhancer'-type activation of mammalian transcription.
 ...
PMID:RNA polymerase II C-terminal domain required for enhancer-driven transcription. 771 9

We have previously shown that stilbene estrogen (diethyl-stilbestrol, DES) covalently binds to nonhistone nuclear proteins both in vivo and in vitro. In this study, we demonstrate the differential effects of DES exposure on in organelle transcriptional activity in nuclei isolated from kidney (target organ of cancer) and liver (non target organ) of hamsters. Kidney RNA polymerase (RNA pol) I and III activities were significantly inhibited by 50% at days 8 and 15 of DES exposure compared to that of controls. Liver RNA pol I and III activities were only modestly inhibited (17 and 22%, respectively) by 2 and 8 days of DES exposure, respectively. However, longer exposure of DES to animals did not produce any significant effects on RNA pol I activity. The activity of RNA pol II was affected by DES exposure in both liver and kidney. DES treatment for two days resulted in an increase in RNA pol II activity in kidney. The enhanced enzyme activity was decreased to 50% of that of the control at 15 days of DES treatment. Unlike RNA pol I and III, RNA pol II activity in the liver was inhibited in a time-dependent fashion in response to DES exposure. To understand the mechanism of transcriptional inhibition by DES, we analyzed the effect of DES exposure on the expression of hepatic RNA pol II at both mRNA and protein levels and also phosphorylation of hepatic RNA pol II. The total amount of transcripts or protein contents of hepatic RNA pol II was not altered in response to DES exposure to hamster for 15 days. Total phosphorylation of hepatic RNA pol II was also not affected by 15 days DES exposure. However tyrosine phosphorylation of hepatic RNA pol II was lowered by 2.8-fold compared to that of control enzyme in response to DES exposure for 15 days. An inhibitory effect of DES on the total RNA polymerase activity in both kidney and liver nuclei in the presence of endogenous template was observed in vitro. No inhibitory effect of DES was observed in vitro on transcriptional activity in the presence of exogenously added DNA template. Based on these data it appears that the in vivo inhibition of transcription by DES may be due to alterations in chromatin template or the level of transcription regulating proteins and not due to decreased availability of the chromatin template and/or RNA polymerase. Whether DES related inhibition of transcriptional activity plays a role in the development of kidney cancer is not clear.
 ...
PMID:Organ-specific inhibition of types I, II and III transcriptional activity in hamsters exposed to stilbene estrogen. 776 59


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>