In vitro termination efficiency change depending on sequence and distance in the elongation path

Abstract

When transcription is carried out employing prokaryotic RNA polymerase, the transcription complex shows a dramatic conformational change during all the steps of transcription. Because this change affects polymerase action on the transcription event, any variation of elongation path will be related to the termination event. To investigate the relationship between termination efficiency and elongation path in the transcription unit, were constructed several transcription units having various distances and sequences between phage T7 $\phi$10 promoter and T$\phi$ terminator. Transcription units with the mutations of sequences affecting transcription initiation step, polymerase binding and promoter melting, decreased sensitively the relative promoter strength but showed little relationship with the termination efficiency. These observations indicate that the termination efficiency is determined by the steps after transcription initiation. Because variation of termination efficiency was observed whenever a region of elongation path before a termination site was deleted by restriction enzyme digestion, transcript lengths as well as template sequences must influence termination efficiency. Transcription units generated by unidirectional deletion from plasmid pYH1 with a promoter and a terminator had 3\% to 83\% efficiency variation. Any variation, even single nucleotide deletion, in promoter-proximal region or terminator-proximal region of transcription unit derived termination efficiency change, as well as the cases with the same distance and different sequence. These results indicate that distance as well as sequence of the elongation path may affect termination efficiency and that elongation step is coupled with termination event. In transcription units having tandem terminators, termination efficiency differed in the first and the second terminators, and termination efficiencies were changed by the deletion in this unit, although this terminator had the same loop f...