TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

Abstract

The Escherichia coli F element-encoded protein TraR is a distant homolog of the chromosome-encoded transcription factor DksA. Here we address the mechanism by which TraR acts as a global regulator, inhibiting some promoters and activating others. We show that TraR regulates transcription directly in vitro by binding to the secondary channel of RNA polymerase (RNAP) using interactions similar, but not identical, to those of DksA. Even though it binds to RNAP with only slightly higher affinity than DksA and is only half the size of DksA, TraR by itself inhibits transcription as strongly as DksA and ppGpp combined and much more than DksA alone. Furthermore, unlike DksA, TraR activates transcription even in the absence of ppGpp. TraR lacks the residues that interact with ppGpp in DksA, and TraR binding to RNAP uses the residues in the β′ rim helices that contribute to the ppGpp binding site in the DksA–ppGpp–RNAP complex. Thus, unlike DksA, TraR does not bind ppGpp. We propose a model in which TraR mimics the effects of DksA and ppGpp together by binding directly to the region of the RNAP secondary channel that otherwise binds ppGpp, and its N-terminal region, like the coiled-coil tip of DksA, engages the active-site region of the enzyme and affects transcription allosterically. These data provide insights into the function not only of TraR but also of an evolutionarily widespread and diverse family of TraR-like proteins encoded by bacteria, as well as bacteriophages and other extrachromosomal elements.

E. coliTraR allosterically regulates transcription initiation by altering RNA polymerase conformation and dynamics

“…TraR is a distant homolog of DksA. Although only half the length of DksA, TraR regulates Eco transcription by binding to the RNAP secondary channel and mimicking the combined effects of DksA and ppGpp (Blankschien et al, 2009;Gopalkrishnan et al, 2017). TraR is encoded by the conjugative F plasmid and is expressed from the pY promoter as part of the major tra operon transcript (Frost et al, 1994;Maneewannakul and Ippen-Ihler, 1993).…”

“…rpsT P2, expressing S20), and activates amino acid biosynthesis and transport promoters (e.g. pthrABC, phisG, pargI, plivJ) in vivo and in vitro (Blankschien et al, 2009;Gopalkrishnan et al, 2017). The affinity of TraR for RNAP is only slightly higher than that of DksA, yet its effects on promoters negatively regulated by DksA/ppGpp in vitro are as large or larger than those of DksA/ppGpp (Gopalkrishnan et al, 2017).…”

“…pthrABC, phisG, pargI, plivJ) in vivo and in vitro (Blankschien et al, 2009;Gopalkrishnan et al, 2017). The affinity of TraR for RNAP is only slightly higher than that of DksA, yet its effects on promoters negatively regulated by DksA/ppGpp in vitro are as large or larger than those of DksA/ppGpp (Gopalkrishnan et al, 2017). The effects of TraR on promoters positively regulated by ppGpp/DksA are also independent of ppGpp (Gopalkrishnan et al, 2017).…”

“…Models for DksA/ppGpp and TraR binding to RNAP have been proposed based on biochemical and genetic approaches (Gopalkrishnan et al, 2017;Parshin et al, 2015;Ross et al, 2013;. Crystal structures of DksA/ppGpp/RNAP and TraR/RNAP confirmed the general features of these models and provided additional detail about their interactions with RNAP, but did not reveal the mechanism of inhibition or activation, in large part because of crystal packing constraints on the movement of mobile regions of the complex (Molodtsov et al, 2018).…”

“…To help understand TraR regulation and principles of the regulation of transcription initiation in general, we used single particle cryo-electron microscopy (cryo-EM) to examine structures of Es 70 alone, Eσ 70 bound to TraR (TraR-Es 70 ), and Es 70 bound to a promoter inhibited by TraR [rpsT P2; (Gopalkrishnan et al, 2017)]. Cryo-EM allows the visualization of multiple conformational states populated in solution and in the absence of crystal packing constraints.…”