Pausing and termination of transcription within the early region of bacteriophage T7 DNA in vitro

Abstract

The process of in vitro RNA chain elongation by Escherichia coli RNA polymerase transcribing the early genetic region of T7 DNA has been studied using a quantitative RNA polymerase assay procedure together with analysis of nascent transcripts by polyacrylamide gel electrophoresis. When transcription is initiated synchronously at the T7 A1 promoter, there is extensive pausing of elongating complexes at specific sites on the DNA as revealed by transient formation of discrete RNA bands detected by gel electrophoresis. This pausing is also signalled by the rapid loss of transcriptional synchrony among the population of elongating polymerase molecules. Although transcriptional pausing is more readily detected at low substrate concentrations, it is still prevalent, even at optimal substrate concentrations, and even at optimal substrate concentrations, RNA polymerase molecules are paused up to 70% of the time during in vitro elongation. Hence, pausing must be reduced substantially to obtain in vivo elongation rates. Transcriptional pause sites are numerous and are distributed throughout the T7 early region. They were not confined to intercistronic regions, nor do we detect strong pause sites located in these regions under a variety of conditions. Pause sites are heterogeneous as judged by differences in the efficiency with which they cause elongating polymerase molecules to pause and in the length of the resulting pause. RNA polymerase responds differently to certain of these sites in the presence of the transcriptional regulatory factors rho-protein and L-protein, raising the possibility that these sites play a role in transcriptional regulation in vivo. The sites at which rho-protein causes RNA chain release in the T7 early region appear to be pause sites. However, not all pause sites are rho release sites and this class includes some of the strongest pause sites. rho release sites are not coincident with the T7 intercistronic regions.