Termination of transcription by Escherichia coli ribonucleic acid polymerase in vitro. Effect of altered reaction conditions and mutations in the enzyme protein on termination with T7 and T3 deoxyribonucleic acids

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Abstract
Both bacteriophage T7 and the related bacteriophage T3 have strong termination sites for bacterial RNA polymerase located near 20% on the standard genome map. These termination sites are used with 90% efficiency in vivo, even in cells which contain a defective .rho. protein. Under normal reaction conditions in vitro, E. coli RNA polymerase terminates with 90% efficiency at the T7 terminator site, but shows little or no termination at the corresponding T3 locus. Thus, the 2 templates form an ideal in vitro test system with which to study the parameters that govern transcriptional termination. Termination at these sites was monitored by following the time course of RNA synthesis under conditions where only a single transcriptional cycle is carried out and by following the size distribution of RNA chains by gel electrophoresis. Termination of the T7 termination site is unaffected by a large variety of changes in reaction conditions, by quantitative cleavage of the nascent RNA during the reaction with a mixture of single- and double-stranded specific ribonucleases, or by a number of different mutations in the subunits of RNA polymerase, including .omega.. Similarly, a large variety of changes in reaction conditions fail to enhance termination at the T3 terminator site, including change in temperature, MgCl2 concentration and glycerol concentration or the addition of dimethyl sulfoxide, ethanol or spermidine to the reaction. In the presence of elevated salt concentrations, at low ribonucleoside triphosphate concentrations, and in the presence of formamide, efficient in vitro utilization of the T3 terminator is seen. Changes in the RNA polymerase protein can also enhance utilization of the T3 site. A class of rifampicin-resistant rpoB mutants was identified which produce a rifampicin-resistant RNA polymerase, which is able to utilize the T3 terminator site in vitro. Similarly, the normal Bacillus subtilis RNA polymerase utilizes the T3 terminator site in vitro with high efficiency.