Using single-molecule fluorescence resonance energy transfer, Richard H. Ebright and co workers have defined bacterial RNA polymerase (RNAP) clamp conformation at each step in transcription initiation and elongation. The researchers find that the clamp predominantly is open in free RNAP and in early intermediates in transcription initiation but closes upon formation of a catalytically competent transcription initiation complex and remains closed during initial transcription and transcription elongation. The researchers show that four RNAP inhibitors interfere with clamp opening. The researchers propose that clamp opening allows DNA to be loaded into and unwound in the RNAP active-center cleft, that DNA loading and unwinding trigger clamp closure, and that clamp closure accounts for the high stability of initiation complexes and the high stability and processivity of elongation complexes.
One broadly significant aspect of the work is the development of powerful methods that enable the incorporation of fluorescent probes at specific sites of interest within megadalton-scale protein complexes.
Determination of RNAP clamp conformation in solution
(A) Measurement of single-molecule FRET between fluorescent probes incorporated at the tips of the RNAP beta' pincer (clamp) and the RNAP beta pincer. Open (red), partly closed (yellow), and closed (green) RNAP clamp conformational states are as observed in crystal structures.
(B) Incorporation of fluorescent probes at the tips of the RNAP beta' pincer (clamp) and the RNAP beta pincer, by unnatural amino acid mutagenesis to incorporate 4-azidophenylalanine at sites of interest in beta' and beta subunits, followed by Staudinger ligation to incorporate fluorescent probes at 4-azidophenylalanines in beta' and beta subunits, followed by in vitro reconstitution of RNAP from labelled beta' and beta subunits and unlabelled alpha and omega subunits.
(C) Relationship between single-molecule FRET efficiencies, E, and RNAP clamp conformational states. The red boxes denote the open (red), closed (green), and collapsed (blue) clamp states observed in this work for RNAP in solution.
Chakraborty A, Wang D, Ebright YW, Korlann Y, Kortkhonjia E, Kim T, Chowdhury S, Wigneshweraraj S, Irschik H, Jansen R, Nixon BT, Knight J, Weiss S, Ebright RH. Science 337: 591-595, 2012