An allosteric mechanism of Rho-dependent transcription termination

Abstract

Rho is the essential RNA helicase that sets the borders between transcription units and adjusts transcriptional yield to translational needs in bacteria1 -3. Although Rho is the first termination factor to be discovered 4 , the actual mechanism by which it reaches and disrupts the elongation complex (EC) is unknown. Here we show that the termination-committed Rho molecule associates with RNA polymerase (RNAP) throughout the transcription cycle, i.e. it does not require the nascent transcript for initial binding. Moreover, the formation of the RNAP-Rho complex is crucial for termination. We show further that Rho-dependent termination is a two-step process that involves rapid EC inactivation (trap) and a relatively slow dissociation. Inactivation is the critical rate-limiting step that establishes the position of the termination site. The trap mechanism depends on the allostericallyinduced rearrangement of the RNAP catalytic center via the evolutionary conserved mobile trigger loop (TL) domain, which is also required for EC dissociation. The key structural/functional similarities, which we found between Rho-dependent and intrinsic (Rho-independent) termination pathways, argue that the allosteric mechanism of termination is general and likely to be preserved for all cellular RNAPs throughout evolution.Rho is the general transcription termination factor in bacteria. Concerted cycles of ATP binding, hydrolysis and release induce conformational changes that pull the nascent transcript through the cavity of the hexamer and induce Rho to translocate along the RNA in the 5′ → 3′ direction [5][6][7][8] . Classical tracking models postulate that, once loaded onto the nascent RNA, Rho catches up to the EC and pulls the transcript away from RNAP and the DNA template [9][10][11] . Extensive biochemical and structural analysis of Rho over the past two decades has explained the molecular details of RNA loading and translocation [5][6][7][8][9][10][11] , but not the actual mechanism of termination. Different models that address this issue have been proposed 9,10,12 ( Supplementary Fig. 1). The majority of these models envisioned RNAP as passive in the termination process. Indeed, simple kinetics, i.e. the speed at which RNAP elongates, is thought to be the major determinant of Rho termination sites, many of which coincide with RNAP pause sites. Although kinetic coupling is certainly an important parameter in controlling the efficiency of Rho termination 13,14 , it does not define the conformational state of the enzyme, which renders the EC susceptible to Rho.To study Rho termination in vitro we constructed two templates containing the T7A1 promoter fused to well-characterized Rho termination sequences: trpt' and aRut14 -16 . We also utilizedCorrespondence and requests for materials should be addressed to E.N. (evgeny.nudler@nyumc.org). * These authors contributed equally to this work.Author Contributions V.E., D.D., and J.W. conducted the experimental work, discussed the results, and commented on the manuscript. E.N. de...

Transcription termination maintains chromosome integrity

“…Rho associates with TEC immediately after the start of transcription, followed by NusA and NusG (13,17). Although the majority of TEC thus include Rho, NusA, and NusG, Rho does not act until translation termination, presumably because ribosomes block Rho access to RNA.…”

“…Termination in Escherichia coli is largely mediated by Rho, an RNAdependent ATPase that terminates transcription promoter-distal to unstructured and untranslated RNA (12,13). The RNA/DNA helicase activity of Rho promotes termination by unwinding RNA/DNA hybrid in the RNAP transcription bubble (14).…”

Modulation of Rho-Dependent Transcription Termination in Escherichia coli by the H-NS Family of Proteins

“…The mechanism by which the architecture of the H-NS scaffold affects the process of Rhodependent termination is not clear. In the kinetic coupling model of Rho action (29), an inefficient Rho can be rendered more effective by slowing the rate of transcription elongation; however, the validity of this model, at least in its simple formulation (29), has recently been questioned since an RNA polymerase mutant with increased elongation rate also exhibited enhanced Rho-dependent termination (21). Moreover, our data reveal no change in RNA polymerase elongation rates in vivo in the rho and nusG strains without or with H-NS⌬64 or overexpressed YdgT, at least on the lacZ gene.…”

“…Rho is an RNAbinding protein and possesses RNA-dependent ATPase activity as well as ATP-dependent 5Ј-3Ј translocase/helicase activity on RNA. Recently, Rho has also been reported to bind RNA polymerase (21). NusG has two domains that bind Rho and RNA polymerase, respectively (13,36).…”

RNA secondary structure-dependent termination of transcription

“…Bacteria developed dedicated hairpins to continue using RRS-dependent termination, but also acquired ρ-dependent termination that rely on an RNA-helicase that displaces the elongation complex. 3 In most modern eukaryotes, transcription duties are divided between 3 RNAPs. Pol III retained RSS-dependent mechanism, given the highly structured nature of its RNA products.…”