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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 specificity of transcription of Euglena gracilis Z chloroplast DNA by chloroplast DNA-dependent RNA polymerase in a transcriptionally active chromosome (Hallick, R.B., Lipper, C., Richards, O.C., and Rutter, W.J. (1976) Biochemistry 15, 3039-3045) has been studied. RNA molecules are both initiated and elongated in vitro. The RNA transcripts have been characterized as to their size, nuclease sensitivity, 5'-terminal oligonucleotides, and coding locus on the chloroplast genome. RNA labeled in vitro at the 5' end with [gamma-32P]ATP was digested with RNase T1, RNase A, and S1 nuclease. The resulting 5'-gamma-32P-oligonucleotides were fractionated by gel electrophoresis. In each case, one or two discrete products were obtained, consistent with initiation in vitro only at defined loci. RNA labeled in vitro with [alpha-32P]ATP or CTP has been hybridized to Southern (Southern, E.M. (1975) J. Mol. Biol. 98, 503-517) transfers of restriction endonuclease fragments of chloroplast DNA. The most abundant in vitro transcripts hybridize to chloroplast DNA fragments coding for 23 S, 16 S, and 5 S rRNAs. Only the coding strands of the rRNA genes are transcribed. Non-rDNA sequences of chloroplast DNA are also selectively transcribed but at much lower levels. The transcriptionally active chromosome has proved to be an ideal biochemical preparation for the study of selective transcription of cell organelle DNA.
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PMID:Selective in vitro transcription of Euglena chloroplast ribosomal RNA genes by a transcriptionally active chromosome. 676 27

We have developed a cell-free system for studying the synthesis of mRNA in mammalian cells. The system consists of a dialyzed and concentrated whole-cell extract derived from HeLa cells, small molecules and cofactors needed for transcription, and exogenously added DNA. Accurate transcription by RNA polymerase II is entirely dependent upon addition of promoter-containing eukaryotic DNA. At optimal DNA and extract concentrations, transcription initiation from the adenovirus serotype 2 late promoter is readily detectable, and specific transcripts over 4000 nucleotides in length are observed. The RNA synthesized in vitro contains the same 5' capped RNase T1 undecanucleotide as does the in vivo transcript. RNA synthesis also initiates accurately at both an early and an intermediate adenovirus promoter site.
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PMID:DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. 693 41

An in vitro transcription system from Candida utilis is described. The template used is a hybrid plasmid containing Saccharomyces cerevisiae CYC1 promoter linked to a synthetic 377-bp G-minus casette (1). In vitro transcriptions are carried out in the presence of RNase. T1. Under these conditions only the transcripts that are resistant to RNase T1 accumulate. Using this protocol, it has been shown that in the absence of cytosolic factors RNA polymerase II (pol II) from C. utilis initiated RNA synthesis randomly. But both C. utilis and S. cerevisiae cell-free extracts could direct pol II from C. utilis to initiate transcription accurately. Results also indicated that the general transcription factors are functionally interchangeable between S. cerevisiae and C. utilis.
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PMID:Accurate transcription initiation by RNA polymerase II from Candida utilis. 798 59

Ternary complexes of RNA polymerase containing the DNA template and nascent RNA are the intermediates in transcript elongation in all cells. We have footprinted the RNA transcript with single-strand-specific ribonucleases in ternary complexes of Escherichia coli RNA polymerase. When complexes are treated with elevated levels of ribonucleases A and T1, the nascent transcript can be cleaved to within 3-4 nucleotides of the 3'-terminus. Ternary complexes containing ribonuclease-cleaved transcripts as short as 3 nucleotides remain stable and active, ensuring that the cleavage occurred within an active ternary complex. However, cleavage by ribonuclease I is restricted, and gives a limited digest product of about 16 nt. At lower concentrations of ribonuclease T1, two regions of partial protection are seen. The first region extends through the first 15-16 nucleotides from the 3'-OH terminus; the second region extends from position 30 out to position 45. We interpret these regions of partial protection as defining two RNA product binding sites on the RNA polymerase that bind the product to the enzyme during elongation. Our results rule out the existence of a stable RNA-DNA hybrid in these ternary complexes of greater than 3 base pairs in length.
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PMID:Structural analysis of ternary complexes of Escherichia coli RNA polymerase: ribonuclease footprinting of the nascent RNA in complexes. 989 Sep 1

Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural minus-strand RNAs (containing any posttranscriptional modification that might have occurred) and have tested them for coat protein binding sites and template activity in an in vitro system with the viral RNA polymerase. The enzyme was prepared by an advanced isolation procedure. All three minus strands had a single non-coded G at their 3' terminus. They were not able to withdraw coat protein subunits from virions as free virion RNAs do. No sites protected by coat protein against ribonuclease T1 degradation were found. Two large T1 oligonucleotides from minus RNA 1 and one from minus RNA 3 were bound by coat protein to Millipore filters. Except for minus RNA 3 which caused a minute amount of full-size plus strand to be synthesized, the minus strands did not function as templates for full-size complementary strands. On the other hand, they gave rise to a number of well-defined shorter products, the synthesis of which was stimulated by the addition of coat protein. These products could not be elongated by a chase treatment and were probably the result of internal initiations. It is concluded that, although posttranscriptional modifications of the template and the presence of coat protein may be necessary factors for plus-strand RNA synthesis, they are certainly not sufficient. Our purified in vitro system needs further sophistication.
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PMID:Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral RNA polymerase. 3'-terminal non-coded guanosine and coat protein are insufficient factors for full-size plus-strand synthesis. 1133 91

Nuclear tRNA genes are transcribed by RNA polymerase III (Pol III) and pre-tRNAs are processed into mature tRNAs via complex processes in the nucleus. We have developed an in vitro Pol III-dependent transcription system derived from tobacco cultured cells, which supports efficiently not only transcription of a variety of plant tRNA genes but also 5'-and 3'-end processing, nucleotide modification and splicing of intron-containing pre-tRNAs. The structures of in vitro transcripts have been confirmed by primer extension analysis and by RNase T1 fingerprinting. The optimal Mg2+ concentration differed for each step so that each reaction can be controlled by adjusting the Mg2+ concentration. At 1 mm Mg2+, only transcription occurs so that pre-tRNAs accumulate. The splicing reaction can be initiated by raising Mg2+ ions (> 5 mm) and enhanced by adding 1 mm hexamminecobalt chloride. Using the optimized system for the Nicotiana intron-containing tRNATyr gene, the precise initiation and termination sites of transcription and the splice sites were determined. The presence of 1 mm NAD+ in the reaction mixture leads to the removal of the 2' phosphate at the splice junction of tRNATyr, demonstrating the activity of a 2'-phosphotransferase in the tobacco nuclear extract. Many modified nucleosides such as m2G, m22G, m1A, phi27 and phi35 are introduced in either of the studied transcripts. As shown in other systems, the conversion of U35 to phi requires an intron-containing substrate.
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PMID:A tobacco nuclear extract supporting transcription, processing, splicing and modification of plant intron-containing tRNA precursors. 1184 97

Viral transcription complexes were isolated from SV40-infected cells and incubated in vitro in the presence of [alpha-32P]UTP to allow elongation of the promoter-proximal RNA up to the attenuation sites. The 94 nucleotide attenuated RNA (spanning nucleotides 243-336) was purified, digested with RNase T1 and fingerprinted. The labeled oligonucleotides were then isolated, digested with RNase T2 and their base composition was determined. Based on these analyses 10 consecutive oligonucleotides, spanning residues 259-336, were identified. As the in vivo synthesized oligonucleotides are unlabeled the junctions between labeled and unlabeled oligonucleotides define the in vivo pause sites of RNA polymerase molecules. The characterization of the 10 radioactive spots and their relative intensities allowed the localization of two in vivo pause sites: one at 13-16 nucleotides downstream from the major initiation site presumably at the initial opening of the DNA helix and the second at approximately 40 nucleotides downstream from the major initiation site, just past a GC-rich region of dyad symmetry. It is postulated that pausing of RNA polymerase molecules in the leader region is an essential process in the control of SV40 late transcription.
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PMID:Pausing of RNA polymerase molecules during in vivo transcription of the SV40 leader region. 1189 23

Here we devise a new method for high-throughput comparative sequence analysis. The developed protocol comprises a homogeneous in vitro transcription/RNase cleavage system with the accuracy and data acquisition speed of matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF MS). In summary, the target region is PCR amplified using primers tagged with promoter sequences of T7 or SP6 RNA polymerase. Using RNase T1, the in vitro transcripts are base-specifically cleaved at every G-position. This reaction results in a characteristic pattern of fragment masses that is indicative of the original target sequence. To enable high-throughput analysis, samples are processed with automated liquid handling devices and nanoliter amounts are dispensed onto SpectroCHIP arrays for reliable and homogeneous MALDI preparation. This system enables rapid automated comparative sequence analysis for PCR products up to 1 kb in length. We demonstrate the feasibility of the devised method for analysis of single nucleotide polymorphisms (SNPs) and pathogen identification.
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PMID:RNase T1 mediated base-specific cleavage and MALDI-TOF MS for high-throughput comparative sequence analysis. 1271 92

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR amplified and in vitro-transcribed 16S rRNA gene (rDNA) was used for the identification of mycobacteria. Full-length 16S rDNA reference sequences of 12 type strains of Mycobacterium spp. frequently isolated from clinical specimens were determined by PCR, cloning, and sequencing. For MALDI-TOF MS-based comparative sequence analysis, mycobacterial 16S rDNA signature sequences ( approximately 500 bp) of the 12 type strains and 24 clinical isolates were PCR amplified using RNA promoter-tagged forward primers. T7 RNA polymerase-mediated transcription of forward strands in the presence of 5-methyl ribo-CTP maximized mass differences of fragments generated by base-specific cleavage. In vitro transcripts were subsequently treated with RNase T1, resulting in G-specific cleavage. Sample analysis by MALDI-TOF MS showed a specific mass signal pattern for each of the 12 type strains, allowing unambiguous identification. All 24 clinical isolates were identified unequivocally by comparing their detected mass signal pattern to the reference sequence-derived in silico pattern of the type strains and to the in silico mass patterns of published 16S rDNA sequences. A 16S rDNA microheterogeneity of the Mycobacterium xenopi type strain (DSM 43995) was detected by MALDI-TOF MS and later confirmed by Sanger dideoxy sequencing. In conclusion, analysis of 16S rDNA amplicons by MS after base-specific cleavage of RNA transcripts allowed fast and reliable identification of the Mycobacterium tuberculosis complex and ubiquitous mycobacteria (mycobacteria other than tuberculosis). The technology delivers an open platform for high-throughput microbial identification on the basis of any specific genotypic marker region.
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PMID:Novel mass spectrometry-based tool for genotypic identification of mycobacteria. 1471 74

In Escherichia coli, 6S RNA functions as a modulator of RNA polymerase sigma70-holoenzyme activity, but its biosynthetic pathway remains uncharacterized. In this study, to further understand the regulatory circuit of 6S RNA biosynthesis for the modulation of Esigma70 activity, we have characterized the biogenesis of 6S RNA. We reveal that there are two different precursors, a long and a short molecule, which are transcribed from the distal P2 and proximal P1 promoter, respectively. Transcription from the P2 promoter is both sigma70- and sigmaS-dependent, whereas, in contrast, P1 transcription is sigma70- but not sigmaS-dependent. Both precursors are processed to generate the 5' end of 6S RNA, and while the long precursor is processed exclusively by RNase E, the short precursor is processed by both RNase G and RNase E. Our data indicate that the switching of the utilization of both sigma factors and endoribonucleases in the biogenesis of 6S RNA would play an essential role in modulating its levels in E.coli.
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PMID:Regulation of 6S RNA biogenesis by switching utilization of both sigma factors and endoribonucleases. 1555 May 66


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