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)

The largest subunit of RNA polymerase II (RNAP II) contains a remarkable region of tandem heptapeptide repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser at its carboxyl terminus. This COOH-terminal domain (CTD) is unphosphorylated in RNAP IIA, extensively phosphorylated in RNAP IIO, and absent in RNAP IIB. The reversible phosphorylation of the CTD has been proposed to be integral to each cycle of transcription from the adenovirus-2 major late promoter. The adenovirus-2 major late promoter, however, may not be a good paradigm for the study of CTD function because in vitro transcription from this promoter is not dependent on the CTD. Previous studies suggest that transcription from the murine dihydrofolate reductase (DHFR) promoter requires the CTD. In an effort to investigate the role of the CTD and its phosphorylation, a RNAP II-dependent reconstituted transcription system specific for the DHFR promoter was established. In this reconstituted system, RNAP IIA, but not RNAP IIB, can transcribe from the DHFR promoter. Furthermore, RNAP IIB does not compete with RNAP IIA for preinitiation complex assembly. These results suggest that the CTD plays a critical role in the recruitment of RNAP II to the DHFR promoter. The analysis of preinitiation complexes assembled on the DHFR promoter indicates that RNAP IIA readily assembles into functional preinitiation complexes in contrast to the inefficient assembly of RNAP IIO. However, transcript elongation is catalyzed by RNAP IIO as demonstrated by the photoactivated cross-linking of nascent DHFR transcripts to subunit IIo. These results indicate that transcription from the DHFR promoter involves the reversible phosphorylation of the CTD and support the idea that RNAPs IIA and IIO have essential but distinct functions.
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PMID:RNA polymerases IIA and IIO have distinct roles during transcription from the TATA-less murine dihydrofolate reductase promoter. 822 67

We have investigated the mRNA expression of 2 human protein tyrosine phosphatases with sequence homology to cytoskeletal proteins, PTPH1 and PTPMEG. Northern-blot analysis of PTPH1 using poly (A)+ RNA from normal human colon tissue showed a low-abundance message of 4.3 kb. Reverse-transcriptase/polymerase-chain reaction (RT-PCR) was therefore used to detect it in a wide variety of cell lines including 9 colorectal, 5 gastric, 5 hepatic and 6 hematopoietic tumor cells. PTPH1 mRNA was not detected only in Colo 320 cells over-expressing c-myc mRNA, among the colorectal cancer cell lines examined. When Colo 320 cells were incubated with 5 mM sodium butyrate for 5 days, PTPH1 mRNA became detectable, concomitant with the marked decrease in the expression level of c-myc mRNA. Moreover, the chromosomal localization of PTPH1 gene was investigated by fluorescence in situ hybridization. Interestingly, PTPH1 gene was mapped to 9q31 where the gene for Gorlin syndrome, a putative tumor suppressor gene, exists.
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PMID:Expression and chromosomal assignment of PTPH1 gene encoding a cytosolic protein tyrosine phosphatase homologous to cytoskeletal-associated proteins. 825 32

The highly conserved Wnt genes belong to a widely distributed family of presumptive signaling molecules that have been implicated not only in the regulation of normal pattern formation during embryogenesis and differentiation of cell lineages, but also in oncogenic events. All of the known vertebrate Wnt genes encode for 38- to 43-kDa cysteine-rich putative glycoproteins, which have features typical of secreted growth factors: a hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence, and 22 conserved cysteine residues whose relative spacing is maintained. In this study, we report the cloning and sequencing of several overlapping cDNAs encoding approximately 4.1 kb of the human homologue of Wnt-5A. The mature protein contained 343 residues (M(r) approximately 38,000 excluding any post-translational modifications) with a > 93% homology to the reported sequences of other Wnt-5A proteins (> 99% homologous to mouse Wnt-5A). This protein maintained certain features--a hydrophobic signal sequence, the Wnt-1 family "signature sequence" (CKCHGvSGSC), and a number of other conserved amino acid residues: 24 cysteine residues, 4 asparagine-linked oligosaccharide consensus sequences, and a tyrosine sulfation site--that have been found in all other Wnt-5A proteins. Reverse transcriptase PCR analysis of RNA from a variety of human embryonic, neonatal, and adult cells and/or tissues showed that human Wnt-5A expression was detected only in neonatal heart and lung. It may be relevant, however, that the 3'-untranslated region contained numerous AT-rich motifs that could be involved in the rapid degradation of mRNA. Finally, using a combination of Southern blotting, PCR amplification, and in situ hybridization, the human Wnt-5A (WNT5A) gene was mapped to chromosome 3p14-p21.
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PMID:Molecular cloning of the human proto-oncogene Wnt-5A and mapping of the gene (WNT5A) to chromosome 3p14-p21. 828 27

The carboxy-terminal domain (CTD) of RNA polymerase II consists of multiple repeats of the unique heptad sequence -(Ser-Pro-Thr-Ser-Pro-Ser-Tyr)- which may interact with DNA through the intercalation of adjacent tyrosine aromatic rings. We have examined details of the interaction of this motif with calf thymus DNA through analysis of peptide analogues that contain (1) an amino-terminal tyrosine which mimics the presence of an adjacent heptad repeat and (2) positively-charged lysine residues which facilitate the initial contact between peptide and DNA. Results of fluorescence experiments, NMR titrations, and viscometric analyses indicate that these peptides bind to the DNA helix through a non-classical intercalation mode involving partial aromatic stacking of the tyrosine rings with the Watson-Crick base pairs.
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PMID:Aromatic stacking and bending of the DNA helix by the individual repeat units of the carboxy-terminal domain of RNA polymerase II. 829 83

When Saccharomyces cerevisiae RNA polymerase (Pol) III transcribes the S. cerevisiae SUP4 tRNA(Tyr) gene, it is obliged to navigate past a large, multi-subunit DNA-bound complex of proteins. We have analyzed individual steps of RNA chain elongation on this gene. Slow steps of transcriptional initiation were by-passed by forming 5'-end-labeled, arrested and precisely positioned transcription complexes. Synchronous resumption of chain elongation by these complexes allowed a single round of RNA synthesis and termination to be analyzed in detail. Results for synthesis at 20 degrees C and 0 degrees C, in the presence of 100 microM and 1 mM ribonucleoside triphosphates (NTPs) are presented. RNA chain elongation through assembled transcription complexes was uneven but relatively rapid: at 20 degrees C with 1 mM NTPs, the fastest RNA chains elongated at an average rate of 29 nucleotides (nt)/second, and the median RNA chains elongated at 21 to 22 nt/second on average. These rates are comparable with a recent measurement of the average rate of chain elongation in vivo by Drosophila RNA polymerase II at 25 degrees C. At 0 degree C, RNA chain elongation rates were, on average, approximately 30-fold slower. Quantitative analysis of the individual steps of RNA chain elongation showed that steps of adding U and A to U-terminated RNA chains tended to be relatively slow, and to be more strongly influenced by nucleotide concentration. Termination of transcription occurred in the sequence T7GT6 (in the non-template DNA strand) and was progressive. Transcripts with five, six and seven U residues were formed, and there was even slow readthrough of the T7 stretch, with GU3 adding rapidly, suggesting that incorporation of a single G into the RNA chain served to reset elongation rates substantially or entirely. Stripping transcription factor (TF) IIIC from transcription complexes did not substantially increase overall RNA chain growth rate, but did diminish pausing at a single site upstream of the boxB binding site of TFIIIC. The TFIIIC-generated delay at this single site was estimated to be only approximately 0.15 to 0.2 seconds at 20 degrees C. Quantitative analysis of RNA chain elongation yielded kinetic parameters for the individual steps of nucleotide addition that were used in computer simulations of RNA chain growth. Elongation modeled as a simple sequence of pseudo-first-order reactions yielded computed RNA chain length distributions that remained relatively synchronous during elongation, while observed chain growth quickly became desynchronized.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Analysis of RNA chain elongation and termination by Saccharomyces cerevisiae RNA polymerase III. 830 83

The analysis of RNA chain elongation by Saccharomyces cerevisiae RNA polymerase (Pol) III in the accompanying paper has been extended by examining the encounter of highly purified RNA polymerase with purified individual transcription factors. Arrested ternary transcription complexes were formed with purified Pol III initiating precisely at the 3' overhanging ends of linear DNA. Transcription factors were then bound to DNA and their effects on individual steps of RNA chain elongation were analyzed. The outcome of the encounter between Pol III and TFIIIC was orientation-specific. For RNA synthesis in the sense direction, with Pol III approaching the obstructing protein from the direction of normal transcription, pure TFIIIC rapidly yielded the way to the advancing polymerase: only a single step of RNA chain elongation was slightly slowed by pure TFIIIC occupying its boxB binding site in the SUP4 tRNA(Tyr) gene. In a complete cell-free fraction, protein binding to this tRNA gene likewise generated a delay of only approximately 0.15 to 0.2 second in executing the same step. Transcription by pure Pol III in the sense direction also dissociated the TFIIIC-SUP4 gene complex. The encounter of Pol III elongating RNA chains in the anti-sense direction with the backside of TFIIIC yielded a different outcome. RNA chain elongation paused extensively six to nine base-pairs beyond the downstream edge of the DNA-binding site of TFIIIC, with a median delay of nine seconds, approximately 50 times longer than in the sense direction. At the height of its effect on RNA chain elongation, the TFIIIC-imposed barrier entrapped the great majority of RNA chains, but their elongation was eventually allowed to continue. In contrast, DNA-bound TFIIIB completely blocked RNA chain elongation in the anti-sense direction. The role of the internal promoter element in transcription by Pol III is discussed in the light of this analysis. The large bulk of TFIIIC, which binds with high affinity to boxB, and also to boxA, is particularly suited to occluding its transcription unit to other proteins. At the same time, TFIIIC makes way for transcription so rapidly that it places no limit on the level of gene activity.
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PMID:Encounters of Saccharomyces cerevisiae RNA polymerase III with its transcription factors during RNA chain elongation. 830 84

A cDNA coding for the beta 4 subunit of murine integrin (m beta 4) has been cloned and sequenced using mRNA from a murine lung carcinoma as the template. The 5' sequence contains two AUG codons, the second of which initiates synthesis of the mature protein. The cDNA sequence has an open reading frame coding for 1748 amino acids (aa), including a signal peptide, cysteine-rich region, serine- and threonine-rich region, transmembrane domain, and a cytoplasmic domain of over 1000 aa. Overall, the deduced m beta 4 aa sequence has 88% identity with the human beta 4 subunit (h beta 4) sequence deduced from the sequence of placental mRNA. Reverse transcriptase-polymerase chain reaction using primers flanking splice sites for two variant forms of h beta 4 transcripts provided evidence for alternate splicing of RNA in the murine spleen and to a lesser extent in the skin, uterus, and thymus but was found at only one of the two alternative sites. Five potential glycosylation sites present in the extracellular domain of h beta 4 are conserved in m beta 4. One tyrosine in the terminal region of the cytoplasmic domain (position 1600) is conserved between m beta 4 and h beta 4 and has the consensus sequence for tyrosine phosphorylation. Finally, a genomic restriction map of m beta 4 shows that the gene is about 40 kb in length. No restriction-fragment length polymorphisms were detected between BALB/c liver and BALB/c lung carcinoma DNA.
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PMID:Sequence of a cDNA encoding the beta 4 subunit of murine integrin. 835 87

The function of a TATA element in RNA polymerase (EC 2.7.7.6) III transcription of a naturally TATA-containing U6 snRNA gene and a naturally TATA-less tRNA gene was probed by transcription and Ty3 transposition analyses. Deletion of the TATA box from a U6 minigene did not abolish transcription and Ty3 integration but changed the positions of initiation and insertion. Insertion of the U6 TATA box at three positions upstream of the TATA-less SUP2 tRNA(Tyr) gene resulted in novel transcription initiation and Ty3 integration patterns that depended upon position of the insertion. Nevertheless, the predominant tRNA gene initiation sites were not affected by insertion of the TATA sequence and remained at a fixed distance from the internal box A promoter element. Insertions of the TATA box upstream of a SUP2 box A mutant affected the level of transcription and restricted the use of upstream start sites, but they neither enhanced the use of TATA-dependent initiation sites nor restored expression to the level of the wild-type gene. We conclude that (i) the U6 TATA box is essential in vivo for correct initiation but not for transcription, (ii) a TATA box does not compensate for a weak box A sequence and so cannot perform equivalently, and (iii) the TATA-binding protein, and probably components of transcription factor IIIB, are present on the target at the time of Ty3 integration.
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PMID:Sites of RNA polymerase III transcription initiation and Ty3 integration at the U6 gene are positioned by the TATA box. 838 58

RNA polymerase II is a multisubunit enzyme composed of two large subunits of molecular weight in excess of 100,000 and a collection of 8-10 smaller subunits. The largest subunit, designated IIa, contains at its carboxyl terminus a highly repetitive domain consisting of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Extensive phosphorylation within this COOH-terminal domain (CTD) gives rise to subunit IIo which has a markedly reduced mobility in SDS-polyacrylamide gel electrophoresis (PAGE) relative to subunit IIa. Recent evidence suggests that RNA polymerase IIA, containing an unphosphorylated CTD, is involved in preinitiation complex assembly, whereas RNA polymerase IIO is involved in elongation. Consequently, CTD phosphorylation is thought to occur after RNA polymerase II has bound to the promoter by a protein kinase that stably associates with the preinitiation complex. We present here the partial purification and characterization of two distinct CTD kinases from a HeLa cell transcription extract. These CTD kinases, designated CTDK1 and CTDK2, are fractionated by chromatography on Mono Q. CTDK1 catalyzes the incorporation of approximately 33 pmol of phosphate/pmol of calf thymus RNA polymerase subunit IIa, almost exclusively on serine. CTDK2 catalyzes the incorporation of approximately 50 pmol of phosphate/pmol of calf thymus subunit IIa, predominantly on serine; appreciable phosphate transfer onto threonine is also observed. Phosphorylation by CTDK2, but not CTDK1, results in a complete mobility shift in SDS-PAGE of subunit IIa to the position of IIo. CTDK1 can utilize ATP, dATP, or GTP as phosphate donor, whereas CTDK2 can utilize only ATP or dATP. The apparent Km for ATP is 30 microM for CTDK1 and 60 microM for CTDK2. CTDK1 and CTDK2 also differ in their protein substrate specificity. CTDK1 phosphorylates casein whereas CTDK2 does not. Neither kinase phosphorylates phosvitin or histone H1 to an appreciable extent. CTDK1 and CTDK2 do not appear to be related to cdc2 kinases as determined by their inability to phosphorylate H1 and their failure to react with antibodies directed against the cdc2 kinase. These results establish that a partially fractionated HeLa transcription extract contains two distinct CTD kinases that differ in their nucleotide requirements and in their patterns of CTD phosphorylation.
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PMID:Partial purification and characterization of two distinct protein kinases that differentially phosphorylate the carboxyl-terminal domain of RNA polymerase subunit IIa. 841 77

A photo-cross-linking method has been used to map the subunits of Saccharomyces cerevisiae RNA polymerase (Pol) III with respect to DNA in binary (preinitiation) and ternary (RNA-elongating) transcription complexes. Transcription factor- and Pol III-containing complexes have been assembled on S. cerevisiae SUP4 tRNA(Tyr) gene probes containing the photoactive nucleotide 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP in different specified positions. Covalent DNA-protein linkages form upon irradiation of these complexes, and the Pol III subunits that are cross-linked to individual positions in the SUP4 tRNA gene have been identified. RNA Pol III cross-linking has been shown to require the box B downstream promoter element of the tRNA gene and the presence of transcription factor TFIIIB. Further proof of specificity has been provided by demonstrating that particular Pol III subunits move out of the range of upstream-placed photoactive nucleotides, and that others move into the range of downstream-placed photoactive nucleotides, as a consequence of initiating and elongating RNA chains. Binding and specific placement of Pol III have also been shown to require both the B' and the B" components of TFIIIB. Nine Pol III subunits are cross-linked from different positions of the SUP4 tRNA gene's nontranscribed strand. In binary transcription complexes, the two largest Pol III subunits are accessible to photo-cross-linking over the entire stretch of the DNase I footprint. The 27- and 34-kDa Pol III subunits are also relatively extended along DNA; its upstream projection makes the 34-kDa subunit a candidate for interaction with TFIIIB, while the 27-kDa subunit is accessible to photo-cross-linking from the leading edge of the Pol III binding site. Several subunits, including the 82- and 53-kDa subunits in binary transcription complexes, are relatively localized in their accessibility to cross-linking. Multiple Pol III subunits are accessible to specific cross-linking from a single photoactive nucleotide in the middle of the transcription bubble of an arrested ternary transcription complex. It is suggested that this precisely placed transcription complex comprises a dynamic ensemble of structural states rather than a single perfectly constrained entity.
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PMID:Orientation and topography of RNA polymerase III in transcription complexes. 842 14


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