Testing the occurrence of forward hyper-translocation during the promoter escape transition

Abstract

The macromolecular process of RNA transcription is composed of three phases, initiation, elongation and termination. At many promoters, RNA polymerase (RNAP) repetitively aborts short RNAs 2 to 15 nucleotides (nts) in length before successfully making the transition to the elongation phase. Mutations in the initial transcribed sequence (ITS) region, from +3 to +10, of Eσ70-dependent T5 N25 promoter lengthen the abortive initiation program, and lead to the formation of the very long abortive transcripts (VLATs) of 16 to 21 nts. Recent published work done in the lab suggests that the VLATs formation may result from forward hyper-translocation movement of RNAP during the escape transition. This project is aimed to test the physical occurrence of forward hyper-transcription by binding a non-cleaving E111Q EcoRI protein roadblock to stall the forward translocating RNAP and thus prevent the formation of VLATs.