Rho-dependent transcription termination: more questions than answers

Abstract

Escherichia coli protein Rho is required for the factor-dependent transcription termination by an RNA polymerase and is essential for the viability of the cell. It is a homohexameric protein that recognizes and binds preferably to C-rich sites in the transcribed RNA. Once bound to RNA, it utilizes RNA-dependent ATPase activity and subsequently ATPase-dependent helicase activity to unwind RNA-DNA hybrids and release RNA from a transcribing elongation complex. Studies over the past few decades have highlighted Rho as a molecule and have revealed much of its mechanistic properties. The recently solved crystal structure could explain many of its physiological functions in terms of its structure. Despite all these efforts, many of the fundamental questions pertaining to Rho recognition sites, differential ATPase activity in response to different RNAs, translocation of Rho along the nascent transcript, interactions with elongation complex and finally unwinding and release of RNA remain obscure. In the present review we have attempted to summarize "the knowns" and "the unknowns" of the Rho protein revealed by the recent developments in this field. An attempt has also been made to understand the physiology of Rho in the light of its phylogeny.

Fig. 1

Functional domains of a Rho protomer of E. coli Rho. The figure shows an open ‘key washer’ like pentameric form of Rho. Each protomer has four defined functional domains: Primary RNA binding domain that can bind to a single stranded DNA molecule as well as single stranded RNA molecule extending from residues 22 to 116; P-loop, for ATP binding and ATPase activity, includes residues from 179-183; Q-loop, from 278-290 and R-loop from 322-326 comprising the secondary RNA binding site of Rho.

Fig. 2

An overview of the steps involved in the termination by Rho. The exact phenomenon of Rho translocation, interaction with elongation complex and finally unwinding and releasing RNA are still ambiguous.

Fig. 3

Events of Rho loading and formation of ATPase competent intermediates (based on Kim and Patel 2001). RNA randomly collides with the Rho molecule forming a dynamic Rho-RNA complex. RNA then wraps around the primary binding site of each of the five protomer of the Rho open complex forming a stable moiety. The RNA subsequently passes towards the center of the open Rho ring making contact with the secondary binding site of the Rho molecule forming a steady complex. Once RNA enters the ‘ring’ of Rho, the open conformation gets closed by a sixth subunit, leading to the formation of a highly stable ATPase competent moiety.

Fig. 4

Translocation models of Rho along the template. Tracking, looping, and tethered tracking models.

Fig. 5

Models to explain Rho’s access to RNA-DNA hybrid and release RNA. A, it spools RNA from a distance without making contact with elongation complex. B, Rho forward translocates the elongation complex to get access to the RNA. C, Rho contact with elongation complex changes the conformation of RNA exit channel.

Fig. 6

Schematic representation of the conserved domains of E. coli Rho through NCBI Blast. a. Rho-factor: Transcription termination factor Rho is a bacterial ATP-dependent RNA/DNA helicase. b. ATPase-fllagellum or Flagellum-specific ATPase/type III secretory pathway virulence-related protein: This group of ATPases are responsible for the export of flagellum and virulence-related proteins. The bacterial flagellar motor is similar to the F₀F₁-ATPase, in that they both are proton driven rotary molecular devices. c. RecA-like NTPases: This family includes the NTP binding domain of F1 and V1 H⁺ATPases, DnaB and related helicases as well as bacterial RecA and related eukaryotic and archaeal recombinases. d. F₁ ATP synthase beta subunit, nucleotide-binding domain: The F-ATPase is found in bacterial plasma membranes, mitochondrial inner membranes and in chloroplast thylakoid membranes. It uses a proton gradient to drive ATP synthesis and hydrolyzes ATP to build the proton gradient. e. V/A-type ATP synthase catalytic subunit A. These ATPases couple ATP hydrolysis to the build up of a H⁺ gradient, but V-type ATPases do not catalyze the reverse reaction. The Vacuolar (V-type) ATPase is found in the membranes of vacuoles, the golgi apparatus and in other coated vesicles in eukaryotes. f. F₁ ATP synthase alpha, central domain. The F-ATPase is found in bacterial plasma membranes, mitochondrial inner membranes and in chloroplast thylakoid membranes g. CSP, Cold shock protein domain; RNA-binding domain that functions as a RNA-chaperone in bacteria and is involved in regulating translation in eukaryotes. Contains sub-family of RNA-binding domains in the Rho transcription termination factor. h. AAA-ATPases associated with a variety of cellular activities. i. ATP synthase alpha/beta family, nucleotide-binding domain. This family includes the ATP synthase alpha and beta subunits the ATP synthase associated with flagella. j. Rho: Transcription termination factor. k. Flagellar biosynthesis/type III secretory pathway ATPase: involved in cell motility and secretion/Intracellular trafficking and secretion. l. AtpA: F₀F₁-type ATP synthase, alpha subunit involved in energy production and conversion. m. NtpA: Archaeal/vacuolar-type H⁺-ATPase subunit A involved in energy production and conversion.