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)

To study the role of post-transcriptional polyadenylation in the mechanism of estrogen action, we measured free nucleoplasmic poly(A) polymerase activity in intact uterine nuclei of immature rabbits at timed intervals after a single intravenous dose of estradiol (20 microgram/kg body weight). Uterine nuclear poly(A) polymerase activity was altered in a biphasic manner by estradiol treatment with maximal activities occurring at 0.5 h and 12 h of steroid administration, at which time periods they were about 2- and 3-fold higher than pretreatment levels, respectively. The later increase in the enzyme activity was totally abolished by a prior cycloheximide (0.5 mg/kg) administration, whereas the initial activation of poly(A) polymerase seemed to occur via mechanism(s) independent of protein synthesis. It thus appears that changes in uterine nuclear poly(A) polymerase closely resemble those previously reported for the activity of RNA polymerase II after estradiol treatment.
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PMID:Early changes in nucleoplasmic poly(A) polymerase activity in immature rabbit uterus after estradiol administration. 625 77

The DNA-dependent RNA polymerase (DdRP) is an essential enzyme for transcription of molluscum contagiosum virus (MCV), a member of the family Poxviridae which replicates in the cytoplasm of the infected cell. Using PCR technology and oligonucleotide primers, corresponding to two conserved domains (RQP[T/S]LH and NADFDGDE) of known largest subunits of eucaryotic and procaryotic DNA-dependent RNA polymerases, the DdRP gene of the genome of molluscum contagiosum virus type 1 (MCV-1) was identified and characterized. The oligonucleotide primers were designed according to the coding usage statistics of known open reading frames of the viral genome. The gene for the largest subunit of DdRP was localized within the DNA sequences of a part of the BamHI DNA fragment A (BamHI/HindIII DNA fragment A8a; 13.5 kbp, 0.454 to 0.525 viral map units) of the MCV-1 genome. The DNA nucleotide sequence analysis of a part (6709 bp) of this DNA fragment revealed the presence of 12 open reading frames (ORFs). It was found that ORF-4 (nucleotide position (NP) 2586 to 6452) and ORF-1 (NP 1192 to 1752) encode two polypeptides comprising 1289 (147 kDa) and 187 (22 kDa) amino acid residues, respectively. The comparative analysis of the amino acid sequences of these ORFs to the amino acid sequences of two subunits (RPO1, 147 kDa and RPO6, 22 kDa) of the DdRP of vaccinia virus revealed high amino acid sequence identity/similarity of about 71.9/21.5% and 46.5/39.6%, respectively. In addition it was found that the putative gene position of ORF-11, which is located on the lower strand between the loci of the ORF-1 and ORF-4 (NP 4256 to 4657, 134 aa, 15 kDa), is similar to the genomic arrangement of the J5L protein of vaccinia virus and L5L of variola virus. The value of amino acid sequence identity/similarity between the product of ORF-11 and the corresponding gene of vaccinia virus (J5L) was found to be 43.2/28.8%. The analysis of the amino acid sequence deduced from ORF-3 (NP 261 to 1289, 343 aa, 40 kDa), which is located upstream from the locus of the RPO6 of the MCV-1 genome, showed significant identity/similarity (47.5/35.7%) to the amino acid sequence of the 40-kDa subunit of the poly(A) polymerase (PAP2) of vaccinia virus. The arrangement of the identified loci of the PAP2, RPO6, ORF-11, and RPO1 of the genome of MCV-1 shows that this particular genomic region of the mollucsum contagiosum virus and vaccina virus is colinear.
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PMID:Identification and properties of the genes encoding the poly(A) polymerase and a small (22 kDa) and the largest subunit (147 kDa) of the DNA-dependent RNA polymerase of molluscum contagiosum virus. 761 82

When expression of the vaccinia virus gene encoding RAP94 (a protein that is associated with the viral multisubunit RNA polymerase and confers transcriptional specificity for early promoters) was repressed, the infectious virus yield was reduced by more than 99%. Nevertheless, intermediate- and late-stage viral gene expression and formation of ultrastructurally mature, membrane-enveloped virions occurred under the nonpermissive conditions. The RAP94-deficient particles contained the viral genome, structural proteins, early transcription factor, and certain enzymes but, unlike normal virions, had low or undetectable amounts of the viral RNA polymerase, capping enzyme/termination factor, poly(A) polymerase, DNA-dependent ATPase, RNA helicase, and topoisomerase. The presence of these viral enzymes in the cytoplasm indicated that RAP94 is required for targeting a complex of functionally related proteins involved in the biosynthesis of mRNA.
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PMID:Targeting of a multicomponent transcription apparatus into assembling vaccinia virus particles requires RAP94, an RNA polymerase-associated protein. 810 1

The cytoplasmic location of vaccinia virus replication and evidence that the multisubunit DNA-dependent RNA polymerase, early and late stage transcription factors, capping and methylating enzymes, and poly(A) polymerase are virus encoded raised the possibility that all of the proteins needed for viral mRNA synthesis are of viral origin. Previous studies showed that four components from infected cells, the viral RNA polymerase and capping enzyme and two factors called vaccinia virus intermediate transcription factors (VITFs) 1 and 2, can reconstitute transcription of vaccinia virus intermediate-stage genes in vitro. Here, we demonstrate that VITF-2 can be isolated from the nuclei of uninfected HeLa cells as well as from the cytoplasm of infected cells. The proteins with VITF-2 activity from uninfected and infected cells cochromatographed and cosedimented, suggesting that they are identical. VITF-2 activity was found in extracts of other uninfected human and monkey cells but not in nonpermissive Trichoplusia ni insect cells or in conditionally permissive rabbit kidney 13 cells. VITF-2 activity was present, however, in a permissive line of rabbit kidney 13 cells that had been stably transfected with the vaccinia virus K1L host range gene. We suggest that the VITF-2 level acts as a gauge of the permissive state of the cell and thereby regulates the length of the early prereplicative phase of the infection.
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PMID:A cellular factor is required for transcription of vaccinia viral intermediate-stage genes. 817 Sep 89

We isolated spontaneous extragenic suppressors of a temperature-sensitive, lethal poly(A) polymerase mutation (pap1-1) in Saccharomyces cerevisiae that restore growth at the restrictive temperature of 30 degrees. Three of five suppressors represent alleles of the PDS2 complementation group. The recessive pds2-1 mutation exerts dominant allele-specific suppression over pap1-1, suggesting a direct functional interaction. The suppressor restores to near normal the steady-state concentrations of various mRNAs and total poly(A) reduced by pap1-1 at 30 degrees. Transcriptional chase experiments detect no reduction in the decay rates of mRNAs in the suppressor strain, suggesting that the restoration of steady-state message levels results from increased stable mRNA synthesis. Molecular cloning shows PDS2 to be allelic to RET1, which encodes the second-largest subunit of RNA polymerase III. We observe alterations in both the length and the steady-state amounts of RNA polymerase III transcripts in pds2-1 strains. Previously identified ret1 alleles do not suppress pap1-1, indicating that the pds2 alleles we isolated represent a specific class of RET1 mutations that suppress pap1-1. Suppression of pap1-1 by mutations in an RNA polymerase III subunit suggests a number of potentially novel interactions between these enzymes.
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PMID:RNA polymerase III defects suppress a conditional-lethal poly(A) polymerase mutation in Saccharomyces cerevisiae. 880 89

Production of messenger RNA in eukaryotic cells is a complex, multistep process. mRNA polyadenylation, or 3' processing, requires several protein factors, including cleavage/polyadenylation-specificity factor (CPSF), cleavage-stimulation factor, two cleavage factors and poly(A) polymerase. These proteins seem to be unnecessary for other steps in mRNA synthesis such as transcription and splicing, and factors required for these processes were not considered to be essential for polyadenylation. Nonetheless, these reactions may be linked so that they are effectively coordinated in vivo. For example, the CTD carboxy-terminal domain of the largest subunit of RNA polymerase II (RNAP II) is required for efficient splicing and polyadenylation in vivo, and CPSF is brought to a promoter by the transcription factor TFIID and transferred to RNAP II at the time of transcription initiation. These findings suggest that polyadenylation factors can be recruited to an RNA 3'-processing signal by RNAP II, where they dissociate from the polymerase and initiate polyadenylation. Here we present results that extend this model by showing that RNAP II is actually required, in the absence of transcription, for 3' processing in vitro.
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PMID:RNA polymerase II is an essential mRNA polyadenylation factor. 973 92

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.
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PMID:Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection. 988 Mar 37

In eukaryotes, polyadenylation of pre-mRNA plays an essential role in the initiation step of protein synthesis, as well as in the export and stability of mRNAs. Poly(A) polymerase, the enzyme at the heart of the polyadenylation machinery, is a template-independent RNA polymerase which specifically incorporates ATP at the 3' end of mRNA. We have solved the crystal structure of bovine poly(A) polymerase bound to an ATP analog at 2.5 A resolution. The structure revealed expected and unexpected similarities to other proteins. As expected, the catalytic domain of poly(A) polymerase shares substantial structural homology with other nucleotidyl transferases such as DNA polymerase beta and kanamycin transferase. The C-terminal domain unexpectedly folds into a compact domain reminiscent of the RNA-recognition motif fold. The three invariant aspartates of the catalytic triad ligate two of the three active site metals. One of these metals also contacts the adenine ring. Furthermore, conserved, catalytically important residues contact the nucleotide. These contacts, taken together with metal coordination of the adenine base, provide a structural basis for ATP selection by poly(A) polymerase.
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PMID:Crystal structure of mammalian poly(A) polymerase in complex with an analog of ATP. 1094 2

J3R, the 39-kDa subunit of vaccinia virus poly(A) polymerase, is a multifunctional protein that catalyzes (nucleoside-2'-O-)-methyltransferase activity, serves as a poly(A) polymerase stimulatory factor, and acts as a postreplicative positive transcription elongation factor. Prior results support an association between poly(A) polymerase and the virion RNA polymerase. A possible direct interaction between J3R and H4L subunit of virion RNA polymerase was evaluated. J3R was shown to specifically bind to H4L amino acids 235-256, C terminal to NPH I binding site on H4L. H4L binds to the C-terminal region of J3R between amino acids 169 and 333. The presence of a J3R binding site near to the NPH I binding region on H4L led us to evaluate a physical interaction between NPH I and J3R. The NPH I binding site was located on J3R between amino acids 169 and 249, and J3R was shown to bind to NPH I between amino acids 457 and 524. To evaluate a role for J3R in early gene mRNA synthesis, transcription termination, and/or release, a transcription-competent extract prepared from cells infected with mutant virus lacking J3R, J3-7. Analysis of transcription activity demonstrated that J3R is not required for early mRNA synthesis and is not an essential factor in early gene transcription termination or transcript release in vitro. J3R interaction with NPH I and H4L may serve as a docking site for J3R on the virion RNA polymerase, linking transcription to mRNA cap formation and poly(A) addition.
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PMID:Interaction between the J3R subunit of vaccinia virus poly(A) polymerase and the H4L subunit of the viral RNA polymerase. 1116 28

The pcnB gene, which encodes the principal poly(A) polymerase of Escherichia coli, promotes 3'-polyadenylation and chemical decay of mRNA. However, there is no evidence that pcnB-mediated mRNA destabilization decreases protein synthesis, suggesting that polyadenylation may enhance translational efficiency. Using in vitro translation by E. coli cell extracts and toeprinting analysis of transcripts encoded by the chloramphenicol acetyltransferase (CAT) and beta-galactosidase genes to investigate this notion, we found no effect of poly(A) tails on protein synthesis. However, we observed that 3'-polyguanylation delayed the chemical decay of CAT mRNA and, even more dramatically, increased the ability of CAT mRNA to produce enzymatically active full-length protein in 30 S E. coli cell fractions. This resulted from interference with the primary mechanism for inactivation of CAT transcript function in cell extracts, which occurred by 3'-exonucleolytic degradation rather than endonucleolytic fragmentation by RNase E. Using bacteriophage T7 RNA polymerase to install poly(G) tails on mRNAs transcribed from polymerase chain reaction-generated DNA templates, we observed sharply increased synthesis of active proteins in vitro in coupled transcription/translation reactions. The ability of poly(G) tails to functionally stabilize transcripts from polymerase chain reaction-generated templates allows proteins encoded by translational open reading frames on genomic DNA or cDNA to be synthesized directly and efficiently in vitro.
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PMID:Effects of 3' terminus modifications on mRNA functional decay during in vitro protein synthesis. 1130


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