Transcription termination by phage T7 RNA poymerase

Abstract

There is one factor-independent transcription terminator, called T7T $\phi$, in phagr T7 genome for the phage-encoded RNA polymerase. Termination efficiency of this intrinsic terminator of 41 bp, surrounded by various DNA sequences, was determined in in vitro transcription reactions including linearized plasmid templates. Although the intrinsic terminator was always directly flanked by its upstream genomic sequence of 70 bp, its in vitro termination effeciency varied from 15\% to 82\%, while experimental error range is below 2\%. The variation of the efficiency depended only on the entire sequence in a transcription unit, while the sequences outside of the unit did not have any effect. In all cases, however, the hairpin RNA structure of the terminator seems to be formed, as predicted by the energy minimization method of RNA folding. Also there seems to be no particular sequences that enhance or reduce the efficiency regardless of their positions. Each termination efficiency did not vary upon the amount of transcripts produced, except in one case. Pulse labeling of transcripts in time course of in vitro tramscription reaction revealed that the efficiency of the terminator in one particular construct (pGEM3ZT), but not in other constructs, was observed high ($\sim$60%) at an early time point but decreased expontially until a low efficiency ($\sim$20\%) persists. Cotranscription of the pGEM3ZT with other templates resulted in reduced termination efficncy from the other templates. In order to identify the trans-acting element in the pGEM3ZT transcription reaction mixtures, after one-hour transcription the T7 promoter was destructed by Hinfl digestion, T7 RNA polymerase and Hinfl were removed by phenol extraction, and then the mixture was included in the second reaction with a second template and newly added polymerase. The resulting reduced termination efficiency of the second templte strongly suggests that the trans-acting element is the RNA transcript from pGEM3Z...