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

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The synthesis of rRNA in the ribosome-free extracts S100 of E. coli cells was about 30 and 60% of total transcript when E. coli DNA and DNA of lambda rifd 18 phage were used respectively. The synthesis of rRNA with both types of DNA was inhibited in 5--6 times by 0.8 mM ppGpp, while the synthesis of total RNA decreased only two-fold. Selective action of ppGpp on rRNA synthesis is due to the intensive inhibition of the initiation of transcription of the appropriate genes. The rRNA synthesis and the inhibitory capacity of ppGpp was shown to dependend on the KCl concentration in S 100 extracts. These results indicate that ppGpp is the main factor controlling rRNA synthesis both in isolated RNA polymerase system and in cell-free extracts.
...
PMID:[The effect of guanosinetetraphosphate on rRNA synthesis in E. coli extracts]. 36 18

A molecular cDNA clone (P1 KIN) was isolated that encodes the human RNA-dependent P1/eIF-2 alpha protein kinase. The complete cDNA sequence of the P1 KIN cDNA was determined; the longest open reading frame (ORF) encoded a 551 amino acid protein with a deduced molecular weight of 62055 Da. Transcripts prepared from the P1 KIN cDNA by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of a protein indistinguishable by immunoprecipitation and immunoblot gel analyses from the authentic 67-kDa P1 protein synthesized in human U cells treated with interferon (IFN). Furthermore, by use of a sensitive primer extension assay with T7 DNA polymerase, the major site of translation initiation within the deduced ORF of the P1 KIN cDNA was directly identified. Northern RNA gel-blot analysis revealed that the P1 KIN cDNA strongly hybridized to two IFN-induced mRNAs present in both human amnion U cells and HeLa cells; their sizes were 2.5 and 6 kb. Both transcripts were efficiently induced by IFN-alpha, but poorly by IFN-gamma. Polyclonal antibody was prepared against the product of the P1 KIN cDNA expressed in Escherichia coli. In Western blot analysis the antibody recognized a 67-kDa protein induced in human cells by IFN-alpha and, in addition, a 90-kDa protein whose level was not greatly altered by IFN treatment. The IFN-induced 67-kDa protein was found associated with the ribosomal salt-wash fraction of IFN-treated human cells, whereas the 90-kDa protein was predominantly in the S100 soluble fraction. The time course for the induction by IFN-alpha of RNA-dependent protein P1 kinase activity measured by immunoprecipitation was comparable to the time course for protein P1 induction measured by Western immunoblot analysis. The amino acid sequence of P1/eIF-2 alpha protein kinase deduced from the cDNA was 62% identical with the 518-residue murine TIK kinase and contained, within the carboxy-terminal half of the protein, the motifs commonly conserved among protein-serine/threonine kinases. The amino-terminal half of the P1 protein did not possess conserved kinase motifs, but did show extensive homology with vaccinia virus-predicted protein E3L.
...
PMID:Mechanism of interferon action: cDNA structure, expression, and regulation of the interferon-induced, RNA-dependent P1/eIF-2 alpha protein kinase from human cells. 137 53

We have reconstituted biologically relevant transcriptional antitermination in vitro by the phage lambda N protein. This required the isolation of NusG, a newly identified Escherichia coli transcription elongation factor. NusG is encoded by an E. coli gene, formerly called U and now called nusG, in which a mutation affects antitermination by N in vivo. Efficient antitermination by N in our reconstituted system depends on the bacterial proteins NusG, NusA, NusB, and ribosomal protein S10 (which functions without ribosomes in transcriptional antitermination). In reactions containing E. coli S100 extract, NusG is stoichiometrically bound to lambda N-modified transcription elongation complexes. We used RNA polymerase affinity chromatography to show that NusG binds to the core component of E. coli RNA polymerase. This binding is weak, and the stable association of NusG with lambda elongation complexes additionally requires at least N, NusA, and the boxA component of an N utilization site. In reactions containing bacterial S100 extract, NusG and NusB are also present in elongation complexes transcribing E. coli boxA-containing rDNA.
...
PMID:NusG, a new Escherichia coli elongation factor involved in transcriptional antitermination by the N protein of phage lambda. 153 77

5 S RNA processing in Drosophila melanogaster, the removal of 15 nucleotides from the 3' end of the 135-nucleotide (nt) primary transcript, may play an important role in the regulation of 5 S RNA transport and ribosome assembly. We have uncoupled processing from transcription using gel purified primary transcripts processed in vitro by a cellular S100 extract. The RNA was generated by a homologous transcription system or by a T7 RNA polymerase reaction using a constructed 5 S RNA gene linked to a T7 promoter. In vitro D. melanogaster 5 S RNA processing is heat- and EDTA-sensitive, suggesting a requirement for protein, and produces a 3' end characteristic of mature 5 S RNA. Processing of substrate RNAs with altered 3' ends shows that the 3'-U5 tail (nt 131-135) inhibits the reaction. 30 nt, including all of domain IV and most of domain V, are dispensible for processing, whereas deletions including the base of stem V and all or part of stem III severely inhibit it. Several possible mechanisms are discussed.
...
PMID:In vitro processing of Drosophila melanogaster 5 S ribosomal RNA. 3' end effects and requirement for internal domains of mature 5 S RNA. 190 21

The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase. At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence. In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence. The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively. Typical transcription reactions yielded 500-700 moles RNA per mole template. This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts. The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100). Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed.
...
PMID:Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA. 219 63

A Schizosaccharomyces pombe U6 small nuclear RNA gene containing an intron has been described. We find that the S. pombe U6 gene is transcribed in a human (HeLa) cell S100 extract with an alpha-amanitin sensitivity characteristic of RNA polymerase III. The S. pombe U6 gene is also transcribed after transfection into human cells. The transcription of vertebrate U6 RNA genes by RNA polymerase III does not require intragenic control elements. The intron of the S. pombe U6 gene disrupts a "box A"-like intragenic sequence that is typically an RNA polymerase III transcription control element. This, together with the transcription of the S. pombe U6 gene by human RNA polymerase III, suggests that it is recognized by human U6 gene-specific transcription machinery.
...
PMID:An intron-containing Schizosaccharomyces pombe U6 RNA gene can be transcribed by human RNA polymerase III. 229 73

We have analysed the transcription of a functional human 7SL gene by RNA polymerase III (RNAPIII) in S100 extracts in vitro. Accurate and efficient synthesis of 7S L RNA depends on the presence of (i) an upstream sequence and (ii) an internal promoter element located within the first 22 bp of the gene. These findings were substantiated by DNase I footprinting. Mutations of the internal promoter identified the doublet CG [nucleotide (nt) +15/+16] outside the A-box homologue (nt +5 to +14) as being essential for both proper promoter function in the in vitro transcription assay and competition in the template-exclusion assay. Fractionation of S100 extracts identified two fractions required in addition to RNAPIII for faithful transcription of the gene. Each of these two fractions gave rise to one of two footprints observed in DNase I protection experiments, indicating that at least two DNA-binding factors are involved.
...
PMID:Sequence and factor requirements for faithful in vitro transcription of human 7SL DNA. 232 74

This paper describes studies of initiation of transcription by RNA polymerase I in vitro. The protocols take advantage of the observation that active transcription complexes precipitate when incubated with S100 extracts. The pellets contain less than 5% of the protein present in unfractionated extracts and are stable to centrifugal washing. This permits rapid manipulation of the reaction conditions and facilitates kinetic studies of aspects of the initiation reaction. An initiated complex has been defined which forms rapidly at 30 degrees C and is associated with formation of the first phosphodiester bond of nascent rRNA. Once formed, initiated complexes are capable of elongation in the presence of heparin or KCl in concentrations sufficient to preclude subsequent initiation. One can therefore estimate the number of initiated complexes formed in a given reaction by measuring the number of full length transcripts recovered in a KCl or heparin-start reaction. The number of such complexes formed correlates well with the formation of a presumptive initiator trinucleotide ApCpU.
...
PMID:Hormonal regulation of transcription of rDNA. Formation of initiated complexes by RNA polymerase I in vitro. 239 51

High-speed supernatant (S100) extracts derived from homogenized Ascaris suum embryos efficiently transcribe added RNA polymerase III templates including cloned 5S rRNA genes of the filarial parasite Brugia malayi. Several criteria, including two-dimensional RNase T1 oligonucleotide fingerprint analysis, indicate that in vitro transcription is accurately initiated and terminated.
...
PMID:Accurate and efficient RNA polymerase III transcription in a cell-free extract prepared from Ascaris suum embryos. 274 46

An in vitro processing system of mouse rRNA was achieved using an RNA polymerase I-specific transcription system, (S100) and recombinant plasmids consisting of mouse rRNA gene (rDNA) segments containing the transcription initiation and 5'-terminal region of 18S (or 41S) rRNA. Pulse-chase experiments showed that a specific processing occurred with transcripts of the plasmid DNAs when the direction of transcription was the correct orientation relative to the 18S rRNA coding sequence, but not with transcripts of the DNA templates in which this coding sequence was in the opposite orientation. From the S1 nuclease protection analyses, we concluded that there are several steps of endonucleolytic cleavage including one 105 nucleotides upstream from the 5' end of 18S rRNA. Intermediates cleaved at this site were identified in in vivo processing of rRNA. This result indicates that endonucleolytic cleavage takes place 105 nucleotides upstream from the 5' terminus of 18S rRNA prior to the formation of mature 18S rRNA. Trimming or cleavage of the 105 nucleotides may be involved in the formation of the 5' terminus of mature 18S rRNA.
...
PMID:Coupled transcription and processing of mouse ribosomal RNA in a cell-free system. 300 77


1 2 3 4 Next >>

---