Single-Molecule Analysis of Transcription

We are using single-molecule fluorescence resonance energy transfer to define fundamental aspects of transcription initiation, elongation, and termination.In published work, we have shown that initial transcription proceeds through a “scrunching” mechanism, in which RNA polymerase (RNAP) remains fixed on promoter DNA and pulls downstream DNA past its active center. We have shown further that putative alternative mechanisms for RNAP-active-center translocation in initial transcription, involving “transient excursions” of RNAP or “inchworming” of RNAP, do not occur. The results support a model in which a stressed intermediate, with DNA-unwinding stress and DNA-compaction stress, is formed during initial transcription, and in which accumulated stress is used to drive breakage of RNAP-promoter interactions during promoter escape.In unpublished work, we are assessing opening and closing of the RNAP active-center-cleft, movements of modules of sigma relative to RNAP in transcription initiation, movements of modules of the RNAP active center in transcription elongation, and movements of RNAP relative to DNA in transcription termination.In further unpublished work, carried out in support of these studies, we have developed reagents and procedures that permit incorporation of a fluorescent probe at any position of interest within a transcription complex.