Contributions of RNA secondary structure and length of the thymidine tract to transcription termination at the thr operon attenuator

Abstract

The thr operon of Escherichia coli is regulated by an attenuation mechanism in which regulated transcription termination occurs in response to the levels of charged tRNAthr and tRNAile. Transcription of the thr operon regulatory region in vitro produces a 162-base transcript that is terminated efficiently at the attenuator. The attenuator sequence is similar to other rho-independent terminators. It contains a G + C region of dyad symmetry followed by a run of 9 A + T residues. We have characterized in detail the sequence requirements for efficient transcription termination in vitro. Using a set of point mutations in the G + C region of dyad symmetry of the thr attenuator, we have characterized the effects of these mutations on the efficiency of transcription termination. The efficiency was reduced in all of the mutants analyzed with the greatest effect being an approximate 20% decrease in termination. In some instances the electrophoretic mobilities of the terminated transcripts on 8% polyacrylamide, 8 M urea gels were shifted substantially relative to the wild type-terminated transcript, but the sites of transcription termination were altered by only a few base pairs. We also constructed a set of deletions removing consecutive thymidines which follow the G + C-rich region of dyad symmetry. Removal of 1 or 3 of the 9 thymidine residues had no effect on termination efficiency in vitro or in vivo. Removal of four to six thymidines caused a linear decrease in the efficiency of termination. When only one or two thymidines were present in the template, termination was completely abolished. These results indicate that both the integrity of the RNA stem and the length of the consecutive thymidine residues are important signals recognized by RNA polymerase during transcription of the thr operon regulatory region.