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

Abstract

Elongation on SV40 DNA F1 by E. coli RNA polymerase was studied, looking specifically at the length of the transcript as a function of time By running the transcription reactions at 18.degree. C with limited enzyme and adding heparin or rifampicin after elongation has started, almost exclusive initiation from the SV40 DNA preferred promoter site was achieved. Within 1500 nucleotides of the initiation 9 prominent sites were observed and a number of minor sites 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 (nucleoside triphosphates), were observed 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.degree. C) used in the experiments since they are also observed at 37.degree. C. The first 4 of these sites were sequenced by using the 3''-O-methyl analogs of the ribonucleotide triphosphates. No sequence homology between the pause sites was found. The kinetics of the pause reactions do not fit a 1st-order model but do correspond to a scheme where continuation through a pause site and termination at a pause site are represented. For 1 of the pause sites, the relaxation time for continuation through the pause site was determined to be .apprx. 2.5 min and for termination .apprx. 50 min at 18.degree. C. If the concentration of 1 of the NTPs is lowered to 10 .mu.M, the strength of a pause site can be increased if that NTP is contained in the pause. 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 sites 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 .cntdot. RNA hybrid is quite stable followed by a region that is very unstable may cause termination. When BrUTP replaced UTP, little effect was observed on the pause sites. The addition of .rho. termination factor causes termination to increase in all the pause sites with a length > 300 nucleotides. In these experiments, those pause sites had continuation relaxation times > 45 s at 37.degree. C. Regardless of the nature of a pause, .rho. probably cause at least some termination at all hesitation sites with a relaxation time > 45 s. All the results are discussed in terms of a kinetic model for the termination of elongation.