Poster abstracts

Poster number 8 submitted by Monali NandyMazumdar

Stabilization of the downstream dsDNA by interaction with the RNA polymerase 'gate' loop

Monali NandyMazumdar (The Ohio State University), Yuri Nedialkov (The Ohio State University), Anastasia Sevostyanova (Yale University), Irina Artsimovitch (The Ohio State University)

Abstract:
RNA polymerase (RNAP) loads the promoter DNA into the main channel, separates the DNA strands to form a transcription bubble, and establishes interactions with the duplex DNA in front of the active site to form a transcriptionally-competent open complex. The β subunit “gate” loop (βGL) was first described as an element lying opposite to the β’ clamp domain of RNAP and was proposed to direct conformational rearrangement of the DNA during open complex formation (1). Our studies on the GL identified it as a key element required for function of RfaH, a non-essential NusG paralogue (2). NusG and its homologs from other organisms establish similar interactions with the βGL and the β’ clamp, locking the RNAP in a closed, processive state. A deletion of the βGL was lethal in Escherichia coli, supporting the essential role of NusG in increasing transcription processivity. Surprisingly, we find that RNAP lacking the GL displays normal elongation, Rho-dependent termination, and response to NusG, and only minor defects in transcript cleavage and termination. By contrast, the deletion of GL has dramatic effects on the structure and stability of open promoter complexes in vitro and on rRNA synthesis in vivo. These observations support the originally proposed function of GL in facilitating the promoter DNA melting.

References:
1) Vassylyev D.G., Sekine S., Laptenko O., Lee J., Vassylyeva M.N., Borukhov S., Yokoyama S. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution. Nature. 2002;417:712–719
2) Sevostyanova A, Belogurov GA, Mooney RA, Landick R, Artsimovitch I., The β subunit gate loop is required for RNA polymerase modification by RfaH and NusG. Mol Cell. 2011; 43(2):253--62

Keywords: gate loop, RNA polymerase, transcription