The β Subunit Gate Loop Mediates Antitermination Modification of RNA Polymerase

Abstract

Synthesis of long RNA chains by multisubunit RNA polymerases (RNAPs) requires accessory proteins that help RNAP bypass numerous roadblocks it encounters along the way. These “antitermination” proteins switch the elongating RNAP into a processive state, but the molecular mechanism of this phenomena remains unknown. Bacterial antiterminator RfaH is an excellent model to address this key mechanistic question because its structure, the mechanism of recruitment and its binding site on RNAP have been determined. RfaH increases expression of the distal genes in operons that have ops DNA elements. We found that RfaH binds simultaneously to the regions of β and β′ subunits that constitutes “jaws” of RNAP, locking it in a closed conformation. Deletion of a conserved β gate loop (GL) has little effect on intrinsic properties of RNAP but completely abolishes its response to RfaH and NusG, a general paralog of RfaH. qRT‐PCR analysis of ops‐containing operon expression driven by wild‐type or ΔGL RNAP in the presence or absence of RfaH confirmed in vitro observations. Finally, we showed also that GL deletion does not compromise stimulation of Rho‐dependent termination by NusG, function that is thought to be independent from AP effect of NusG and based on physical tethering of Rho to the elongation complex. We propose that NusG‐like proteins restrict conformational mobility of RNAP, thereby locking it in a processive state.