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At least 199 records · Page 11Linked to original sources
PubMed1981

Escherichia coli deoxyribonucleic acid dependent ribonucleic acid polymerase transcriptional pause sites on SV40 DNA F1.

We have studied elongation of SV40 DNA F1 by E. coli RNA polymerase looking specifically at the length of the transcript as a function of time. By running the transcription reactions at 18 degrees C with limited enzyme and adding heparin or rifampicin after elongation has started, we have achieved almost exclusive initiation from the SV40 DNA preferred promotor size [Zain, B. S., Weissmann, S. M., Lebowitz, P., & Lewis, A. M., Jr. (1973) J. Virol. 11, 682-693]. In the region within 1500 nucleotides of the initiation we observe nine prominent sites and a number of minor site where hesitation during elongation occurs. The positions of these hesitation points or pause sites are not effected by changes in the salt concentration, the simultaneous lowering of the concentrations of all the NTPs, or by increases in the RNA polymerase concentration, implying that the pause sites are a consequence of the RNA, DNA, and RNA polymerase ternary complex. The pause sites are not an artifact of the lowered temperature (18 degrees C) used in the experiments since they are also observed at 37 degrees C. The first four of these sites have been sequenced by using the 3'-O-methyl analogues of the ribonucleotide triphosphates. We have found no sequence homology between the pause sites. The kinetics of the pause reactions do not fit a first-order model but do correspond to a scheme were continuation through a pause site and termination at a pause site are both represented. For one of the pause sites, the relaxation time for continuation through the pause site was determined to be approximately 2.5 min and for the termination approximately 50 min at 18 degrees C. If the concentration of one of the NTPs is lowered to 10 muM, the strength of a pause site can be increased if that NTP is contained in the pause. Also, minor pause sites are observed at regions in the RNA sequence which are rich in the NTP that has the lowered concentration. When GTP is replaced by ITP during transcription, a new set of pause site quite different from the normal sites of hesitation are observed. The major new pause sites occur at or near sequences in the RNA which are rich in I-U residues preceded by a region rich in C residues. This indicates, as has been previously noted, that sequences where the DNA.RNA hybrid is quite stable followed by a region that is very unstable may cause termination. When BrUTP replaced UTP, very little effect was observed on the pause sites. The addition of p termination factor causes termintion to increase in all the pause sites with a length greater than 300 nucleotides. In the type of experiments performed here, those pause sites had continuation relaxation times greater than 45 s at 37 degrees C. This implies that regardless of the nature of a pause, p will cause at least some termination at all hesitation sites with a relaxation time greater than 45 s. All the results are discussed in terms of a kinetic model for the termination of elongation.

Base Sequence↗
PubMed1982

Independent binding sites in mouse 5.8S ribosomal ribonucleic acid for 28S ribosomal ribonucleic acid.

Limited digestion of mouse 5.8S ribosomal RNA (rRNA) with RNase T2 generates 5'- and 3'-terminal "half-molecules". These fragments are capable of independently and specifically binding to 28S rRNA, so there exist at least two contacts in the 5.8S rRNA for the 28S rRNA. The dissociation constants for the 5.8S/28S, 5' 5.8S fragment/28S, and 3' 5.8S fragment/28S complexes are 9 x 10(-8) M, 6 x 10(-8) M, and 13 x 10(-8) M, respectively. Thus, each of the fragment binding sites contributes about equally to the overall binding energy of the 5.8S/28S rRNA complex, and the binding sites act independently, rather than cooperatively. The dissociation constants suggest that the 5.8S rRNA termini from short, irregular helices with 28S rRNA. Thermal denaturation data on complexes containing 28S rRNA and each of the half-molecules of 5.8S rRNA indicate that the 5'-terminal binding site(s) exist(s) in a single conformation while the 3'-terminal site exhibits two conformational alternatives. The functional significance of the different conformational states is presently indeterminate, but the possibility they may represent alternative forms of a conformational switch operative during ribosome function is discussed.

Animals↗
PubMed1980

Conformational mobility of deoxyribonucleic acid, transfer ribonucleic acid, and poly(adenylic acid) as monitored by carbon-13 nuclear magnetic resonance relaxation.

The molecular motion of DNA, the native form of tRNA, and partially denatured poly(A) has been investigated by carbon-13 nuclear magnetic resonance (13C NMR). The nuclear Overhauser effect of the RNA samples was measured at 25.1 and 50.3 MHZ, and the spin-lattice relaxation time of all the samples was measured at 50.3 MHZ. The NMR data indicate that the local motion of the ribose carbons is much less restricted than that of the bases for DNA and tRNA. The local motion correlation times of the ribose carbons are in the range of 1-7 ns for the samples investigated. The local motion correlation times of the different nucleic acids are quite similar with the exception that the 2' carbon of DNA and poly(A) is apparently less restricted than that for tRNA. The local motion correlation times of the ribose carbons, except perhaps the 2', do not appear to be strongly coupled to the conformation of the polynucleotide. The 13C NMR results can be combined with those of other investigations to obtain a consistent picture of the internal and overall motions of polynucleotides which have a backbone that is much more flexible than that of the bases.

Carbon Isotopes↗
PubMed1977

In vitro transcription of Moloney leukemia virus genes in infected cell nuclei and chromatin: elongation of chromatin associated ribonucleic acid by Escherichia coli ribonucleic acid polymerase.

The in vitro transcription of viral specific DNA sequences in nuclei and chromatin isolated from mouse cells chronically infected with Moloney murine leukemia virus (Mo-MuLV) has been studied. The in vitro RNA synthesized by Escherichia coli RNA polymerase has been isolated by sulfhydryl affinity column following reaction in the presence of 5-mercuriuridine triphosphate. By comparison of the Crt curves of the in vitro RNA with that of 70S viral RNA, the content of viral sequences is found to be 1.3% in nuclei product and 0.24% in chromatin product which is lower than the 2.5% found in chromatin associated RNA. This latter value, however, is very close to the in vivo viral RNA content in pulse-labeled [3H]RNA of the infected cells. Unexpectedly, it is observed that over 20% of the chromatin associated RNA prelabeled in vivo with [5-3H]uridine is elongated and tagged with Hg atoms during RNA synthesis catalyzed by the exogenous E. coli RNA polymerase in the presence of Hg-UTP. The elongation reaction is dependent on the presence of all four nucleotide triphosphates and appears to be due to E. coli RNA polymerase per se. It is suggested that most of the viral specific sequences observed in the in vitro RNA products are very likely initiated and derived from the chromatin associated species. The implication of the present findings for in vitro RNA synthesis in nuclei and chromatin as related to regulation of gene expression is discussed.

Animals↗