Transcription termination mechanism of class I signal for Bacteriophage T7 RNA Polymerase

Abstract

A hairpin-dependent termination site which is classified as Class I signal recognized by bacteriophage T7 RNA polymerases consists of the formation of a secondary structure of a stable stem-loop followed by runs of UTP in the nascent RNA. Interactions between these particular elements together trigger the termination process. Such kind of terminator, e.g. T7-TΦ, relies on different termination mechanism compared to the sequence specific termination which is usually called Class II signal. Previously, we have studied sequence specific termination mechanism which involves the conserved sequence (CS). In this study, we followed the kinetic pathway of transcription on E.coli rrnB T1 terminator and observed the sequential pausing character along the terminator upstream the termination site. In addition, we also figured out the elongation competency near the termination site. Elongation complexes of T7 RNA polymerase were stopped at desired positions by stepwise walking. Then we obtained the specific TECs and evaluated their stability via the percentage of retained RNA transcript against time-course. Different terminators were tested and found that the significant abrupt drop of stability took place around two to five base pair upstream the termination site, based on the terminator character, and the release rate of RNA in TEC increased further near the point of termination site. This phenomenon is discrepant to the transcription termination by E.coli RNAP which indicated that all events, including release of RNA transcript, loss of contact of RNA to polymerase and melting of DNA-RNA hybrid happened just at the point of termination site. Therefore, all our data suggested that Class I termination mechanism by T7 RNAP maybe come down to another way- the sequential changes in elongating complex before termination.