Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro.

Publisher Website
Google Scholar
Abstract
Termination of transcription by Escherichia coli RNA polymerase in vitro appears to depend primarily on 2 structural features of the termination site; a G + C(guanine, cytosine)-rich region of dyad symmetry and a series of terminal uridine residues in the transcript. To determine whether these 2 features are sufficient to specify .rho.-independent termination in vitro, new sequences were introduced within a tryptophan (trp) operon structural gene to create 2 sites with these characteristics. Transcription with wild-type RNA polymerase in vitro demonstrates that discrete termination occurs at one of these new sites, although at a low level. Use of the mutant RNA polymerase rpo203, which is more sensitive to certain weak terminators than is the wild-type enzyme, increases termination at both sites. The activity of our synthetic terminators was compared with those of several termination sites in the E. coli trp operon. Under normal conditions of transcription in vitro, termination becomes more efficient with an increase in the length of the stem in the RNA hairpin or an increase in the number of consecutive uridine residues. Transcription with the rpo203 polymerase and with ribonucleotide analogs gives changes consistent with these general trends. A model for termination involving separate but essential roles for the RNA hairpin and the stretch of uridines in the transcript is supported.