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 describe a fast and robust new assay format to measure poly(A) polymerase (PAP) activity in a microtiter plate format. The new assay principle uses only natural nucleotide triphosphates and avoids a labour-intensive filtration step. A coupled enzymatic system combining PAP and reverse transcriptase forms the basis of the assay. The PAP generates a poly(A) tail on a RNA substrate and the reverse transcriptase is used to quantify the polyadenylated RNA by extension of a biotinylated oligo-dT primer. We demonstrate the principle of the assay using influenza virus RNA polymerase and yeast PAP as examples. A specific increase in the K(m) value for ATP and the observation of burst kinetics in the polyadenylation dependent, but not in the polyadenylation independent, assay suggest that a rate limiting step of influenza polymerase activity occurs after transcription elongation. Yeast PAP was used to validate the assay as an example of a template independent PAP. The new yeast PAP assay was approximately 100-fold more sensitive than the conventional TCA precipitation assay for yeast PAP, but the kinetic analysis of the PAP reaction gave similar results in both assays. The two enzymes show important differences with respect to inhibition by 3'-deoxy-ATP. Whereas the K(i) value for 3'-deoxy-ATP (105-117 microM) is similar to the K(m) value for ATP (186 microM) in the case of influenza RNA polymerase, the K(i) value for 3'-deoxy-ATP (0.4-0.6 microM) is approximately 100-fold lower than the K(m) value for ATP (50 microM) in the case of yeast PAP.
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PMID:A sensitive, single-tube assay to measure the enzymatic activities of influenza RNA polymerase and other poly(A) polymerases: application to kinetic and inhibitor analysis. 1143 13

PAP (polyadenylate polymerase) is the template-independent RNA polymerase responsible for synthesis of the 3' poly(A) tails of mRNA. To investigate the role of proton transfer in the catalytic mechanism of PAP, the pH dependence of the steady-state kinetic parameters of yeast PAP were determined for the forward (adenyl transfer) and reverse (pyrophosphorolysis) reactions. The results indicate that productive formation of an enzyme-RNA-MgATP complex is pH independent over a broad pH range, but that formation of an active enzyme-RNA-MgPPi complex is strongly pH dependent, consistent with the production of a proton on the enzyme in the forward reaction. The pH dependence of the maximum velocity of the forward reaction suggests two protonic species are involved in enzyme catalysis. Optimal enzyme activity requires one species to be protonated and the other deprotonated. The deuterium solvent isotope effect on Vmax is also consistent with proton transfer involved in catalysis of a rate-determining step. Finally, pKa calculations of PAP were performed by the MCCE (multiconformational continuum electrostatic) method. Together, the data support that the protonation of residues Lys215 and Tyr224 exhibit co-operativity that is important for MgATP2- and MgPPi2- binding/dissociation, and suggest these residues function in electrostatic, but not in general acid, catalysis.
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PMID:Proton transfer in the mechanism of polyadenylate polymerase. 1928 52

In this work, we describe RapA-dependent polyadenylation of model RNA substrates and endogenous, RNA polymerase-associated nucleic acid fragments. We demonstrate that the Escherichia coli RNA polymerase obtained through the classic purification procedure carries endogenous RNA oligonucleotides, which, in the presence of ATP, are polyriboadenylated in a RapA-dependent manner by an accessory poly(rA) polymerase. RNA polymerase isolated from poly(A) polymerase- (PAP-) and polynucleotide phosphorylase- (PNP-) deficient E. coli strain lacks accessory (rA)(n)-synthetic activity. Experiments with reconstituted RNA polymerase-PAP and RNA polymerase-PNP mixtures suggest that RapA enables the polyadenylation by PAP of RNA polymerase-associated RNA. Mutations disrupting RapA's ATP-hydrolytic function disrupt RapA-dependent polyadenylation, and the rapA(-)E. coli strain displays a measurable reduction in RNA polyadenylation. RapA-dependent polyadenylation can also be modulated by mutations in the section of RapA's SWI/SNF domain linked to interaction with single-stranded nucleic acid. We have developed enzymatic assays in which model, synthetic RNAs are polyriboadenylated in a RapA-dependent manner. Taken together, our results are consistent with RapA acting as an RNA polymerase-associated, ATP-dependent RNA translocase. Our work further links RapA to RNA remodeling and provides new mechanistic insights into the functional interaction between RNA polymerase and RapA.
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PMID:RapA, Escherichia coli RNA polymerase SWI/SNF subunit-dependent polyadenylation of RNA. 2129 17

Drosophila Trf4-1 (DmTrf4-1) is a polyadenylation polymerase or terminal nucleotidyl transferase (PAP/TENT) that has been reported to add poly(A) tails to snRNAs in nucleus or mRNAs in cytoplasm. Here, we found that the loss of Trf4-1 resulted in the reduction of mRNAs and primary miRNAs (pri-miRNAs) in both Drosophila S2 cells and adult flies. Interestingly, the role of Trf4-1 in transcription is independent of its PAP/TENT activity. Moreover, using the chromatin immunoprecipitation assay, we uncovered that the loss of Trf4-1 led to abnormal RNA polymerase II accumulation and reduced H3K4me3 binding in promoter regions. Thus, our study indicates a positive role of Trf4-1 in the transcription of mRNAs and pri-miRNAs.
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PMID:Drosophila Trf4-1 involves in mRNA and primary miRNA transcription. 3083 53