Transcription termination by bacteriophage T3 and SP6 RNA polymerases at Rho-independent terminators.

Abstract

Transcription termination of T3 and SP6 DNA-dependent RNA polymerases have been studied on the DNA templates containing the threonine (thr) attenuator and its variants. The thr attenuator is from the regulatory region of the thr operon of Escherichia coli. The DNA template, encoding the thr attenuator, contains specific features of the rho-independent terminators. It comprises a dG + dC rich dyad symmetry, encoding a stem-and-loop RNA, which is followed by a poly(U) region at the 3'-end. Thirteen attenuator variants have been analyzed for their ability to terminate transcription and the results indicated that the structure as well as the sequence in the G + C rich region of RNA hairpin affect termination of both RNA polymerases. Also, a single base change in the A residues of the hairpin failed to influence termination, whereas changes in the poly(U) region significantly reduced the termination of both T3 and SP6 RNA polymerases. The requirement of a poly(U) region for termination by T3 and SP6 RNA polymerases was studied with nested deletion mutants in this region. The minimum number of U residues required for termination of SP6 and T3 RNA polymerases was five and three, respectively. However, both RNA polymerases needed at least eight U residues to reach a termination efficiency close to that achieved by wild-type thr attenuator encoding nine U residues. In addition, the orientation of the loop sequences of the RNA hairpin did not affect the transcription termination of either of the bacteriophage RNA polymerases.