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J Microbiol. Author manuscript; available in PMC 2007 March 28.
Published in final edited form as:
J Microbiol. 2006 February ; 44(1): 11–22.
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Rho-dependent Transcription Termination: More Questions than
Answers
Sharmistha Banerjee, Jisha Chalissery, Irfan Bandey, and Ranjan Sen*
Laboratory of Transcription Biology, Center for DNA Fingerprinting and Diagnostics, ECIL Road,
Nacharam, Hyderabad-500076, India.
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.
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Keywords
Rho; transcription termination; RNA polymerase; RNA-dependent ATPase; RNA/DNA helicase
The regulated expression of genes in an organism confides on the signals in the DNA that
effect every phase of the transcription process by a DNA dependent RNA polymerase. Once
the process of transcription begins, the RNA polymerase makes a stable elongation complex
with the DNA and the nascent RNA (Wilson and von Hippel, 1994; Mooney et al., 1998).
The regulation of gene expression begins from appropriate recognition of the promoter site
by a RNA polymerase and extends to each step during RNA chain elongation when the
transcription is interrupted by a pause, an arrest or a termination signal. Thus, initiation,
elongation and termination of the transcription act in concert for an orderly expression of
genes. Distinguished by their mechanism and structural features there are two kinds of
terminators in E. coli genome. (a) Intrinsic terminators characterized by a GC rich inverted
repeat followed by a stretch of consecutive thymidylate that induce RNA polymerase to
pause, destabilize and disengage for releasing RNA without the involvement of any
auxiliary proteins and (b) the factor-dependent terminators that have a highly inconsistent
sequence homology but characteristically require the essential protein factor Rho for
termination (for review on transcription in prokaryotes see Richardson, 1993; Richardson
and Greenblatt, 1996; Uptain et al., 1997).
*
To whom correspondence should be addressed. (Tel) 91-40-27151344; (Fax) 91-40-27155610 (E-mail) rsen@cdfd.org.in.
, Banerjee et al. Page 2
Rho, encoded by the gene rho, is a universal protein found throughout the bacterial world
with a very few exceptions. Rho is a homohexamer with a protomer of 46.8 KDa which is a
product of a single polypeptide of 419 residues. Since its discovery and purification by J.W.
Roberts in 1969 (Roberts, 1969), Rho has become instrumental in exploring the regulatory
role of auxiliary proteins in prokaryotic transcription termination by RNA polymerases.
In the cell, Rho binds to untranslated naked RNAs and terminates the synthesis of mRNA at
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the end of a significant number of operons. Rho is essential for the survival of most of the
prokaryotes. As a molecule, Rho is a RNA/DNA helicase or translocase that dissociates
RNA polymerase from DNA template to release RNA, deriving energy by hydrolyzing ATP
through its RNA-dependent ATPase activity to bring about termination (Richardson, 2002;
Richardson, 2003). As transcription and translation occur simultaneously in prokaryotes,
when translational termination occurs within a gene, Rho can cause transcriptional
termination of the downstream gene in an operon, thus preventing its expression. This effect,
called translational polarity (Adhya and Gottesman, 1978; Nudler and Gottesman 2002), can
be suppressed by either a mutation in rho or by its inhibitor (Das et al., 1976; Linderoth and
Calendar, 1991).
Properties of Rho is a reflection of its structure
Functional domains
Prior to the elucidation of the crystal structure of Rho complexed with nucleic acid in free
and nucleotide (AMPPNP) bound states (Skordalakes and Berger, 2003), attempts were
made to dissect the domain structure by cleavage protection assays, partial tryptic digestion,
photo affinity labeling with nucleotides and synthetic ribo oligos and NMR and X-ray
studies of N-terminal RNA binding domain (Bear et al., 1985; Dolan et al., 1990; Bogden et
al., 1999; Wei and Richardson, 2001a). These studies, along with the crystal structure and
further mutational analysis have specified the following functional domains in a Rho
protomer (Fig. 1): (a) Core RNA binding domain called the primary RNA binding site of
Rho that can bind to a single stranded DNA molecule as well as a single stranded RNA
molecule held responsible for tethering of Rho to the Rho loading or Rho utilization (rut)
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site, extends from residues 22-116 (Modrak and Richardson, 1994). (b) P-loop, associated
with ATP binding and ATPase activity of Rho, spanning from 179-183 is highly conserved
among RecA family of ATPase (Opperman and Richardson, 1994; Wei and Richardson,
2001a). (c) Q-loop and R-loop comprising the secondary RNA binding site of Rho. Q-loop
is formed by 8-residue segment within 278-290 (Wei and Richardson, 2001a; Wei and
Richardson, 2001b; Xu et al., 2002; Skordalakes and Berger, 2003). R-loop, a less defined
and characterized part of Rho protein, is thought to span between 322-326 amino acid
residues in E. coli (Miwa et al., 1995; Burgess and Richardson, 2001a; Skordalakes and
Berger, 2003).
The Oligomeric assembly of Rho
Rho can self assemble in a solution with a variety of assembly states, the homohexamer
being the most predominant (Geiselmann et al., 1992a). The major factors that influence
Rho subunit assembly are the ionic environment, concentration of the Rho protein and the
presence of cofactors (Geiselmann et al., 1992a; Geiselmann et al., 1992b). Rho protomers
assemble into stable dimeric or tetrameric forms under a high concentration of salt, low
protein concentration and in the absence of cofactors. Almost a homogenous hexameric
population of Rho can be obtained in a weak ionic environment of 50-100 mM salt and a
2-10 μM of Rho, either in presence or absence of a small RNA cofactor (Geiselmann et al.,
1992a). The hexameric unit of Rho in the absence of RNA can exist in either an open ‘key
washer’ like pentameric form or in closed ‘ring like’ hexameric form. Both have been
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, Banerjee et al. Page 3
argued to be the functional states of Rho and the ‘open’ Rho structure can be converted into
closed form in the presence of a single stranded nucleic acid that is presumed to wrap
around the periphery of the Rho molecule, often in presence of nucleotide cofactors (Gan
and Richardson, 1999; Yu et al., 2000). A dodecamer state has been observed in the
presence of a RNA cofactor with a stoichometry of one Rho hexamer for every two to three
RNA molecules. The dodecamer assembly is speculated to increase the ‘effector
concentration’ of Rho at the site of action in vivo (Geiselmann et al., 1992a). The
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combinatorial analysis through electron microscopic, hydrodynamic, X-ray and neutron
scattering techniques, the geometry of the Rho hexamer was found to be a regular hexagon
(Gogol et al., 1991; Geiselmann et al., 1992a; Geiselmann et al., 1992b).
The crystal structure of Rho revealed its asymmetric nature. Subunit interactions occur
between the connecter loop of the second and the third α-helices in the N-terminal domain
of one subunit with the ‘linker’ region of the other subunit. The C-terminal contact between
subunit is adjacent to the P-loop of the ATP binding domain. The periphery of the ring is
defined by the N-terminal, while the Q-loop of each protomer together contributes to the
constricted inner ring. The movement of one protomer with respect to the other by ∼ 15°
gives rise to displacement of 45° between the first and the last protomer of the pentameric
open ring assembly. This generates a gap sufficient to bind a single molecule of single
stranded nucleic acid (Skordalakes and Berger, 2003). Thus, it supports the interpretation
from earlier studies that ‘notched’ or open ring assembly is the functional state of the protein
for loading RNA into the hole of the hexamer (Yu et al., 2000).
Primary nucleic acid binding site
N-terminal domains consist of three α-helices appended to five stranded β-barrel comprising
the oligonucleotide or oligosaccharide-binding OB fold. Even though earlier experiments
gave direct and indirect evidences of primary RNA binding domain extending from 22-116
(Modrak and Richardson, 1994), the first nucleotide base packs into the hydrophobic pocket
of Tyr80, Glu108 and Tyr110 (Skordalakes and Berger, 2003). Protection assays by a
homonucleotide and by the natural template λ cro containing rut site supported by X-ray
crystallography evidence have revealed that there are three distinct contact regions housed in
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the (i) N-terminal residues 58-62 with Leu58 and Phe62 creating a hydrophobic shelf for
ribose interactions (Bogden et al., 1999), (ii) the loop connecting third and fourth β strands
(Wei and Richardson, 2001a), (iii) residues 103-110, that include the hydrophobic enclosure
of Glu108, Arg109and Try110 engaged in Vander Waals interaction with the base of
cytidine (Wei and Richardson, 2001a). While this enclosure is enough to accommodate
pyrimidines, it is too small for larger molecules like purines. This has been argued to be one
of the reasons for the preference for ‘pyrimidine’-rich sequence for Rho loading. Further X-
ray crystal structure of N-terminal domain has revealed conjunction between Arg66, Glu56
and Asp78 that creates a stearic conformation to select pyrimidines over purines and it is
interpreted that Watson/Crick-like-interaction (stacking of nucleotide bases on aromatic
protein side chains) selects cytosine over uracil (Bogden et al., 1999). This explains from the
structural point of view the basis of recognition of Rho utilization site (rut site) that are rich
in unpaired cytidylate (C) residues and lack secondary structure (Galluppi and Richardson,
1980; Richardson and Richardson, 1992; Wang and von Hippel, 1993). Interestingly the
crystal structure has overcome the notion that primary binding RNA cleft residues are at the
periphery of the Rho molecule. The orientation of the N-terminal modulate primary RNA
binding cleft face inwards, thus making mRNA to follow a somewhat zig-zag path that
extends from the periphery to the center of each protomer for binding to the Rho molecule.
J Microbiol. Author manuscript; available in PMC 2007 March 28.
