Functions of bacteriophage T7 transcription terminator

Abstract

Bacteriophage T7 terminator Tφ is a class I intrinsic terminator recognized by T7 RNA polymerase and defined by a hairpin structure followed by a stretch of five to eight uridines at the 3’ end of RNA transcript. Owing to their simplicity, T7 RNA polymerase and terminator Tφ have been used as models for extensive studies on mechanisms of transcription initiation, elongation and termination. However, studies on physiological roles of terminator Tφ, on the other hand, are somewhat neglected. Here, I want to revisit this well-studied terminator to fill in the seemingly neglected information on its physiological functions during the course of T7 phage infection. In addition to that, a closer look at the critical functional parts of terminator Tφ itself will further expand our knowledge on the mechanism and regulation of this terminator that can be applicable to other intrinsic terminators. The first part of this study focused on dissecting essential functional elements of terminator Tφ, encompassing hairpin stem, hairpin loop and U-stretch. Tφ’s hairpin stem was gradually deleted to examine termination efficiency of truncated variants. I discovered that only the bottom six base pairs of Tφ’s hairpin stem (here named Stem3) were essential for termination function of this terminator. Stem3, however, was the minimal functional hairpin stem for Tφ, as any further single-base pair deletion from this stem resulted in drastic reduction of termination efficiency. Exchanges of Tφ’s hairpin loop to shorter tri-loop and tetra-loop altered termination efficiency to a certain extent, though no significant increase was observed. Shortening the U-stretch following Tφ’s hairpin by even just one uridine residue lead to significant reduction of termination efficiency, suggesting the full-length U-stretch is required for Tφ functionality. Interestingly, double deletion of one base pair from hairpin stem and one uridine from U-stretch resulted in reduction of termination efficiency e...