Investigating the transcription initiation mechanism of E. coli RNAP by FRET (743.1)

Abstract

E. coli RNA Polymerase (RNAP) recognizes the ‐10 and ‐35 upstream elements of the promoter DNA, which bends and wraps around RNAP on the back of the β subunit of the RNAP core. The recognition between RNAP and DNA promoter initiates the melting of about 13bp of DNA duplex surrounding the transcription start site in the active site of RNAP to form a promoter‐RNAP open complex (RPo). Even though transcription of DNA plays a critical role in gene expression and regulation, many important questions about the transcription initiation mechanism remains unanswered. To study the wrapping of DNA around RNAP, we have performed equilibrium FRET experiments on a strong promoter, λPr with Cy3 and Cy5 fluorophores on the upstream and downstream ends of the promoter DNA. We find that wrapping is present in closed complexes, as seen from complexes at 2°C and it persists in open complexes at 19°C. We further confirmed that the fluorophore‐labeled DNA complexed with RNAP is transcriptionally competent. Single‐labeled constructs also show permanganate reactivity, proving that fluorophore‐labeled promoter‐RNAP complexes are open. Further experiments are underway to study the kinetics of wrapping of promoter DNA around RNAP.Grant Funding Source: NIH GM103061