scholarly journals Characterization of double-stranded ribonucleic acid sequences present in the initial transcription products of rat liver chromatin

1977 ◽  
Vol 165 (2) ◽  
pp. 237-245 ◽  
Author(s):  
E Pays
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rat Liver ◽  
Rna Sequences ◽  
Complementary Sequences ◽  
Exogenous Rna ◽  
Liver Chromatin ◽  
Low Ionic Strength

At low ionic strength and with a low exogenous RNA polymerase/DNA ratio, rat liver chromatin directs the synthesis in vitro of RNA sequences rich in double-stranded segments. All the transcripts contain at least one double-stranded sequence. Most of the double-stranded segments are formed by intramolecular base-pairing of inverted complementary sequences separated by a single-stranded loop. They are heterogeneous in size, 35-45% of them being more than 80 nucleotides long. They contain 61-64% G+C, whether synthesized by rat liver RNA polymerase (form B) or Escherichia coli RNA polymerase. The largest double-stranded sequences are found in the largest transcripts, and are the most thermostable. The fidelity of base-matching is better in double-stranded transcripts synthesized on rat liver chromatin by homologous polymerase than in those synthesized on it by a bacterial polymerase, or in those synthesized by either of the two polymerases on pure DNA.

scholarly journals Transcription of rat liver deoxyribonucleic acid in vitro at low ionic strength

1978 ◽  
Vol 175 (1) ◽  
pp. 1-13 ◽  
Author(s):  
E Pays
Keyword(s):  
Rna Polymerase ◽  
Rat Liver ◽  
Chain Elongation ◽  
Rna Sequences ◽  
Double Stranded Dna ◽  
Low Concentrations ◽  
Dna 2 ◽  
Low Ionic Strength ◽  
Dna Ends

1. When RNA polymerase is in excess over DNA, the single-stranded breaks of DNA can be recognized as initiation sites for the ezyme. On the other hand stabel initiation complexes (resistant to inhibition by heparin) are the most abundant under these conditions. The formation of these complexes needs double-stranded DNA. It seems that RNA sequences rich in cytidine are preferentially synthesized; since rat liver DNA is A + T-rich, the transcription thus appears not to be random with respect to the base composition of DNA. 2. When the template is in excess over the polymerase, the single-stranded gaps of DNA are preferentially transcribed by rat liver RNA polymerase B and native DNA regions by Escherichia coli RNA polymerase. 3. With a large excess of DNA over the polymerase, the enzyme activity is markedly inhibited. This inhibition is proportional to the concentration of double-stranded DNA ends, but it also depends on the presence of a contaminant of DNA, removed when DNA is banded in a CsCl gradient. This contaminant could be polyphosphates. Low concentrations of spermine completely reverse this inhibition, by enhancing the rate of RNA chain elongation. 4. Double-stranded RNA is synthesized in great abundance when RNA polymerase is in excess over native DNA. Besides a majority of symmetrical sequences, stable ‘hairpins’ can be found. Whereas the synthesis of symmetrical sequences is more prevalent in polymerase excess, it seems that the proportion of stable ‘hairpins’ in RNA is independent of the polymerase/DNA ratio.

scholarly journals Function-Based Selection and Characterization of Base-Pair Polymorphisms in a Promoter of Escherichia coli RNA Polymerase-ς70

2001 ◽  
Vol 183 (9) ◽  
pp. 2866-2873 ◽  
Author(s):  
Jian Xu ◽  
Barbara C. McCabe ◽  
Gerald B. Koudelka
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Promoter Sequence ◽  
Kinetic Properties ◽  
Optimal Sequence ◽  
Sequence Requirements ◽  
Better Than

ABSTRACT We performed two sets of in vitro selections to dissect the role of the −10 base sequence in determining the rate and efficiency with which Escherichia coli RNA polymerase-ς70forms stable complexes with a promoter. We identified sequences that (i) rapidly form heparin-resistant complexes with RNA polymerase or (ii) form heparin-resistant complexes at very low RNA polymerase concentrations. The sequences selected under the two conditions differ from each other and from the consensus −10 sequence. The selected promoters have the expected enhanced binding and kinetic properties and are functionally better than the consensus promoter sequence in directing RNA synthesis in vitro. Detailed analysis of the selected promoter functions shows that each step in this multistep pathway may have different sequence requirements, meaning that the sequence of a strong promoter does not contain the optimal sequence for each step but instead is a compromise sequence that allows all steps to proceed with minimal constraint.

scholarly journals CHANGES IN CHROMATIN TEMPLATE ACTIVITY AND THEIR RELATIONSHIP TO DNA SYNTHESIS IN MOUSE PAROTID GLANDS STIMULATED BY ISOPROTERENOL

1972 ◽  
Vol 55 (3) ◽  
pp. 554-562 ◽  
Author(s):  
Anna M. Novi ◽  
Renato Baserga
Keyword(s):  
Parotid Gland ◽  
Rna Polymerase ◽  
Dna Synthesis ◽  
Salivary Glands ◽  
Parotid Glands ◽  
Template Activity ◽  
Exogenous Rna ◽  
Chromatin Template ◽  
Liver Chromatin

Chromatin template activity of mouse parotid glands increases after a single injection of isoproterenol (IPR), a procedure that causes, after a lag period of 20 hr, a marked stimulation of DNA synthesis and cell division in salivary glands of rodents. The increase in chromatin template activity occurs as early as 1 hr and peaks between 6 and 10 hr after IPR, paralleling previously reported changes in the incorporation of uridine-3H into total cellular RNA of mouse parotids. Template activity was measured in vitro in a system in which parotid gland chromatin was incubated with an exogenous RNA polymerase isolated from Escherichia coli. Similar results were obtained when template activity of parotid gland chromatin was assayed using an homologous RNA polymerase from mouse liver. Chromatin template activity in mouse parotids was also studied after the administration of drugs capable of inducing in salivary glands both DNA synthesis and secretion or secretion alone. The results indicate that the increased chromatin template activity occurring 6 hr after IPR is related to the subsequent onset of DNA synthesis. Furthermore, the increased chromatin template activity caused by IPR is inhibited by the previous administration of puromycin, an inhibitor of IPR-stimulated DNA synthesis.

III. Transcription Chromatin transcription with mercurated nucleotides

1978 ◽  
Vol 283 (997) ◽  
pp. 379-380 ◽  
Keyword(s):  
Escherichia Coli ◽  
Present Report ◽  
Embryonic Liver ◽  
Globin Mrna ◽  
Rna Sequences ◽  
E Coli ◽  
Nucleotide Triphosphates ◽  
Liver Chromatin ◽  
Cdna Hybridization

Recently, several authors have described the isolation of transcripts synthesized in vitro from chromatin templates with Escherichia coli RNA polymerase using mercury substituted nucleotide triphosphates (Biessman, Gjerset, Levy & McCarthy 1976; Smith & Huang 1976; Crouse, Fodor & Doty 1976). The advantage of this technique is that the mercurated RNA can be separated from contaminating endogenous RNA sequences present in the isolated chromatin by chromatography on sulphydryl Sepharose. The presence of endogenous RNA has previously presented problems when attempting sequence analysis of the in vitro transcripts by cDNA hybridization (Gilmour, Windass, Affara & Paul 1975; Wilson et al . 1975). In the present report mouse embryonic liver chromatin (an erythropoietic tissue, 14 days in utero ) was incubated with E. coli polymerase as previously described (Gilmour & Paul 1973) and with Hg-UTP prepared according to the method of Dale & Ward (1975). Mercurated transcripts were isolated by the use of sulphydryl Sepharose as described in the above paper and the presence of globin mRNA sequences determined by hybridization with [ 3 H]globin cDNA. In separate experiments it was shown that between 80 and 90 % of the newly synthesized RNA is recovered from the incubation.

scholarly journals The use of rat liver nucleoplasm for the characterization of heterogeneous nuclear ribonucleic acid synthesis in vitro

1978 ◽  
Vol 176 (3) ◽  
pp. 715-725 ◽  
Author(s):  
T J C Beebee
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rat Liver ◽  
Dna Sequences ◽  
Acid Synthesis ◽  
Rna Molecules ◽  
Liver Nuclei

1. A nucleoplasmic fraction rich in endogenous RNA polymerase II activity was isolated from rat liver nuclei and conditions were determined under which elongation of RNA molecules initiated in vivo continued at maximal rates in vitro. 2. Elongation rates in vitro were calculated to be about 0.25 nucleotide/s and there were about 7 × 10(3) RNA molecules in the process of being elongated by form-II RNA polymerase per original nucleus. 3. Evidence was obtained suggesting that transcription-dependent release of RNA polymerase II molecules from the template occurred during the incubations in vitro. 4. The nascent RNA was tightly associated with protein and banded as ribonucleoprotein in caesium salt gradients. 5. RNA molecules labelled in vitro were up to 13000 nucleotides in length, but consisted of long unlabelled chains transcribed in vivo with only short labelled sequences added in vitro, and without significant polyadenylation. 6. Hybridization of transcripts in the presence of a vast excess of DNA demonstrated that both form-II RNA polymerase and another enzyme, resistant to low alpha-amanitin concentrations, were synthesizing RNA molecules complementary to both reiterated and unique DNA sequences in the genome.

Characterization of the RNA transcribed in vitro from native mammalian DNA by Escherichia coli RNA polymerase

1976 ◽  
Vol 454 (3) ◽  
pp. 469-479 ◽  
Author(s):  
Angel Alonso ◽  
G.D. Birnie ◽  
L. Kleiman ◽  
A.J. Macgillivray ◽  
John Paul
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase
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