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Rho factor is a hexameric ring-shaped helicase which terminates transcription in Escherichia coli. Two recent crystal structures of Rho in complex with nucleic acid reveal how this helicase ring loads onto mRNA and encircles it.
We present evidence that the transcription termination factor Rho is autogenously regulated in Escherichia coli. The steady-state level of Rho is increased approximately tenfold in rho mutant cells. In the rho+ revertants, the content of Rho is similar to the wild-type level. A rho-/rho+ merodiploid produces equimolar amounts of the mutant and the wild-type Rho polypeptides, both at a reduced level compared to the mutant. The steady-state level of rho messenger RNA is also increased in a rho mutant. A rho-galK transcriptional fusion produces at least tenfold more galactokinase in a rho- strain than in a rho+ strain. In vitro, in a coupled transcription-translation system, the synthesis of Rho protein is specifically inhibited by wild-type Rho but not by Rho15 mutant protein. Anti-Rho antibody specifically stimulates Rho synthesis in the rho+ extract but not in a rho- extract. We suggest that the autogenous regulation of Rho involves premature transcription termination within the rho gene. Regulation of Rho level may provide the cell a mechanism to modulate the expression of genes which are separated from their promoters by Rho-dependent termination signals.
Transcription termination factor rho was purified to near homogeneity from the wild type and temperature-sensitive rho-111 mutant strains of Salmonella typhimurium. Each protein had identical physical properties with respect to native and subunit molecular weight, elution from ion-exchange columns, and poly(C)-dependent ATPase specific activity at 30 degrees C. The mutant protein exhibited a thermolabile poly(C)-dependent ATPase activity. The transcription termination and nascent RNA-dependent ATPase activities associated with the purified wild type S. typhimurium rho protein were not present in the mutant protein. Binding studies demonstrated that the stability of the rho-111:poly(C) complex was significantly more sensitive to ionic strength and temperature than that of the rho +: poly(C) complex. This result suggests that the altered activities of the mutant protein are due to its decreased ability to participate in a specific interaction with RNA which is insensitive to ionic strength. The rho-111 mutation resulted in a 20- to 30-fold elevation in the level of the mutant protein, indicating that rho biosynthesis in S. typhimurium is autogenously regulated. Therefore, defective molecular interactions between the mutant rho protein and RNA might account for the absence of transcription termination in vitro, and the polarity suppressor phenotype and defective autogenous regulation of rho biosynthesis in vivo.
Escherichia coli nusD strains are bacteria that carry mutations in rho, the gene for transcription termination factor Rho, that block the growth of phages T4 and lambdar32. We have identified the rho mutation in six independent nusD strains, and although five of the strains have different mutations, with one exception the mutations are in the proposed RNA-binding domain of Rho. We overexpressed, purified, and characterized the five different mutant Rho proteins. All show substantial RNA-dependent ATPase activity with several homoribopolymers or the lambda cro message as cofactor. At the lambda tR1 Rho-dependent terminator in vitro, all mutant Rho proteins show decreased termination compared with wild-type, and all also terminate within cro at a new terminator, tRE, with endpoints 5' to tR1 at 170, 200, 245 and 260 nucleotides 3' from the transcription start. The mutant Rho proteins are proposed to interfere with bacteriophage T4 growth through indirect effects on host gene expression.
We have investigated the effect of the rho-115 mutation on the catalytic properties of the Escherichia coli termination protein, rho. Comparison of the primary and secondary polynucleotide binding sites activities reveals dramatic differences between the mutant and wild-type molecules. Wild-type rho must bind single-stranded polynucleotides to activate its nucleotide triphosphatase (NTPase) activity, and either poly(C), or poly(dC) plus oligo(C), will suffice. In contrast, attempted activation of the rho-115 NTPase with poly(C) in the presence of poly(dC) showed the latter to be a potent inhibitor. Inclusion of small oligonucleotides such as oligo(C) in the activation assay does not inhibit the poly(C)-induced NTPase reaction of either wild-type rho or rho-115. This would indicate, in the two polynucleotide binding site model for rho proposed by Richardson (Richardson, J.P. (1982) J. Biol. Chem. 251, 5760-5766), that the mutation in rho-115 affects the primary polynucleotide binding site. Transcription termination in vitro at the rho-dependent site trp t' showed dramatically reduced termination with rho-115 protein compared to wild-type rho. In the presence of rho-115, the transcript is longer and termination occurs over a narrower range of nucleotides than with wild-type rho. This suggests that the primary polynucleotide binding site is important not only for efficient termination of transcription but may also be involved in determining the terminal end point of the transcript itself.
Escherichia coli transcription termination factor rho is an RNA-dependent ATPase, and ATPase activity is required for all its functions. We have characterized the binding of ATP to the physiologically relevant hexameric association state of rho in the absence of RNA and have shown that there are six ATP binding sites per rho hexamer. This stoichiometry has been verified by a number of different techniques, including ultracentrifugation, ultrafiltration, and fluorescence titration studies. We have also shown that ATP can bind to isolated monomers of rho when the hexamer is dissociated with the mild denaturant myristyltrimethylammonium bromide, demonstrating that each promoter of rho carries an ATP binding site. The six binding sites that we observe in the rho hexamer are not equivalent; the hexamer contains three strong (Ka approximately 3 x 10(6) M-1) and three weak (Ka approximately 10(5) M-1) binding sites for ATP. The binding constant of the weak binding site is just the reciprocal of the enzymatic Km for ATP as a substrate; thus these weak sites, as well as the strong sites, can, in principle, take part in the catalytic cycle. The asymmetry induced (or manifested) by ATP binding reduces the symmetry of the rho hexamer from a D3 to a pseudo-D3 state. This "breakage" of symmetry has implications for the molecular mechanism of rho, because an asymmetric structure can lead to directional helicase activity by invoking directionally distinct RNA binding and release reactions (see Geiselmann, J., Yager, T.D., & von Hippel, P.H., 1992c, Protein Sci. 1, 861-873).
Transcription termination factor rho from Escherichia coli is a ring-shaped homohexamer of 419 amino acid subunits and catalyzes an ATP-dependent release of nascent RNA transcripts. Previous chemical cross-linking studies suggested that the rho hexamer might have D3 symmetry with three isologous dimers as protomers. However, our recent mutational analysis of rho alongside its putative structural homology to F1-ATPase rather argued for C6 symmetry. To resolve this discrepancy, we have re-investigated the pattern of cross-linking of rho using various cross-linkers with different functional groups and spacer lengths. Upon reaction with dimethyl suberimidate followed by SDS-polyacrylamide gel electrophoresis, rho protein generated a series of cross-linked oligomers up to hexamers, of which dimers migrated as distinct doublet bands of approximately equal intensities. However, the lower band became much stronger than the upper one with dimethyl adipimidate and difluorodinitrobenzene, and vice versa with disuccinimidyl glutarate, disuccinimidyl suberate and disulfosuccinimidyl tartarate. Furthermore, the trimeric products also produced doublet bands, whose relative intensities were again variable with cross-linkers, but in an inverse correlation with those of the dimer bands. These results combined with theoretical considerations support a C6 symmetry model in which cross-linking is assumed to occur stochastically at one of two alternative sites within each subunit interface with variable relative frequencies depending on cross-linkers. The D3 symmetry is excluded, for the putative trimeric subspecies should always retain mutually equal intensities in that case. Detailed inspections of the cross-linking kinetics further revealed a moderate characteristic of C3 symmetry for the rho hexamer such that the collective as well as relative rates of cross-linking at the two available sites could fluctuate between alternating interfaces. The final model designated as C3/6 is also compatible with other functional and structural properties known for rho.
Transcription factor Rho of Eschericia coli is a ring-shaped homohexameric protein that terminates transcripts by its action on nascent RNAs. To test the functional importance of the phylogenetically highly conserved residues of the Q-loop region, four mutant Rho proteins, S281A, K283A, T286A and D290A, were isolated and analyzed for their biochemical properties. All four proteins were very defective in terminating transcripts in vitro at the bacteriophage lambda tR1 terminator and had corresponding defects in ATP hydrolysis activated by lambda cro RNA. Although the four proteins were normal or near normal in their sensitivity to cleavage with H(2)O(2) in the presence of Fe-EDTA and in their ability to bind to lambda cro RNA and ATP, they were defective in RNA-specific, secondary site interactions. This was indicated by the lack of protection from cleavage at their Q-loops by oligo(C) in the presence of poly(dC), and their defects in ATP hydrolysis activated by oligo(C) in the presence of poly(dC). This evidence, together with the observations that cleavage of the Q-loop residues is protected specifically by RNA, suggests that the Q-loop makes interactions with RNA that are essential for activation of ATP hydrolysis and the termination of transcription.
Under approximately physiological conditions, the transcription termination factor rho from Escherichia coli is a hexamer of planar hexagonal geometry [Geiselmann, J., Yager, T. D., Gill, S. C., Calmettes, P., & von Hippel, P. H. (1992) Biochemistry (preceding paper in this issue)]. Here we describe studies that further define the quaternary structure of this hexamer. We use a combination of chemical cross-linking and treatment with mild denaturants to show that the fundamental unit within the rho hexamer is a dimer stabilized by an isologous (or pseudoisologous) bonding interface. Three identical dimers of rho interact via a second type of isologous bonding interface to yield a hexamer with C3 or D3 symmetry. Cross-linking and denaturation experiments definitely rule out C6 and C2 symmetry for the rho hexamer. Data from fluorescence quenching, lifetime, and energy transfer experiments also argue against C2 symmetry. The simplest symmetry assignment that is not contradicted by any experimental data is D3; thus we conclude that the rho hexamer has D3 symmetry. We also consider the positioning of the binding sites for RNA and ATP relative to the coordinate reference frame of the D3 hexamer. Fluorescence energy transfer data are presented and integrated with data from the literature to arrive at a self-consistent model for the quaternary structure of the rho hexamer.
Escherichia coli transcription termination factor Rho shows a 30-fold faster rate of ATP hydrolysis when all three catalytic sites are filled with ATP than when only a single site is filled (Stitt, B. L. and Xu, Y. (1998) J. Biol. Chem. 273, 26477-26486). To study the structural requirements of the substrate for this catalytic cooperativity, rapid mix/chemical quench experiments using various ATP analogs were performed. The results indicate that it is the configuration of the beta- and gamma-phosphoryl groups of ATP that is of primary importance for the rate enhancement. Our results also show that there are kinetically slow branches of the enzyme mechanism that are not seen when the chemistry step of the catalytic cycle is fast. These branches become prominent, however, when two of the three Rho active sites are empty or bear non-hydrolyzable compounds. A first-order step that is slow compared with V(max) catalysis enables a single ATP molecule bound in any one of the three Rho active sites to be hydrolyzed and defines the kinetically slow branches. This first-order step could be a protein conformation change or a rearrangement of bound RNA. The results reinforce the importance of catalytic cooperativity in normal Rho function and suggest that several protein conformations exist along the catalytic pathway.
The Escherichia coli rho transcription termination factor terminates select transcripts and rho activity requires Mg(2+). We investigated whether divalent metal ions other than Mg(2+) catalyze rho-dependent ATP hydrolysis to ADP and P(i) in vitro. The effects of 11 divalent metal ions (Be(2+), Ca(2+), Cd(2+), Co(2+), Cu(2+), Hg(2+), Mn(2+), Ni(2+), Sr(2+), VO(2+), Zn(2+)) on ATPase activity were determined in the absence and presence of MgCl(2). Without MgCl(2), Ca(2+), Cd(2+), Co(2+), Cu(2+), Hg(2+), Mn(2+), Ni(2+), VO(2+), and Zn(2+) activated ATP hydrolysis with either hyberbolic (Ca(2+), Co(2+), Cu(2+), Hg(2+), VO(2+)), peak velocity (Cd(2+), Mn(2+), Zn(2+)), or sigmoidal (Ni(2+)) rate acceleration curves. Sr(2+) was found to be a nonactivator and Be(2+) an inhibitor of rho-dependent ATPase activity. The metals' effects were compared with Mg(2+) and gave different rank orders when either the velocity (V(max), V(peak)) or the efficiency (V(max)/K(M), V(peak)/K(M)) of ATP hydrolysis was used as the determinant (V: Mg(2+) approximately Mn(2+) > Zn(2+) > Co(2+) > Ni(2+) approximately Cd(2+) > Ca(2+) > Cu(2+) > Hg(2+) approximately VO(2+); V/K(M): Mg(2+) > Mn(2+) > Ca(2+) > Co(2+) > Zn(2+) > Cu(2+) > Ni(2+) > Hg(2+) > Cd(2+)). Mg(2+) proved to be the most effective divalent metal. We observed that the metal-dependent rates were affected by metal ion interactions with rho, RNA, and the buffer constituents. Significantly, replacement of the octahedral Mg(2+) ion by metals that typically prefer coordination spheres less than six (Cd(2+), Co(2+), Ni(2+), VO(2+), Zn(2+)) led to ATPase activity, suggesting that the putative Mg x ATP(2-) coordination sphere in rho does not need to remain fully intact for ATP hydrolysis.
The rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho-dependent termination sites on the DNA template, and (in a series of steps that are still largely undefined) brings about transcript termination at these sites. In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional form of rho. We use CD spectroscopy and analytical ultracentrifugation to determine the binding interactions of rho with RNA ligands of defined length ([rC]n where n > or = 6). Rho binds to long RNA chains as a hexamer characterized by D3 symmetry. Each hexamer binds approximately 70 residues of RNA. We show by ultracentrifugation and dynamic laser light scattering that, in the presence of RNA ligands less than 22 nucleotide residues in length, rho changes its quaternary structure and becomes a homogeneous dodecamer. The dodecamer contains six strong binding sites for short RNA ligands: i.e., one site for every two rho protomers. The measured association constant of these short RNAs to rho increases with increasing (rC)n length, up to n = 9, suggesting that the binding site of each rho protomer interacts with 9 RNA nucleotide residues. Oligo (rC) ligands bound to the strong RNA binding sites on the rho dodecamer do not significantly stimulate the RNA-dependent ATPase activity of rho. Based on these features of the rho-RNA interaction and other experimental data we propose a molecular model of the interaction of rho with its cofactors.
The ATPase and helicase activities of the Escherichia coli transcription termination protein rho have been studied under a variety of reaction conditions that alter its transcription termination activity. These conditions include KCl, KOAc, or KGlu concentrations from 50 to 150 mM and Mg(OAc)2 concentrations from 1 to 5 mM (in the presence of 1 mM ATP). In higher KCl or higher Mg(OAc)2 concentrations we found that the translocation of rho hexamers along RNA was slower and less processive than the same process measured at 50 mM monovalent salt concentrations and 1 mM Mg(OAc)2. The ATPase activity of rho was also decreased under reaction conditions that slowed translocation. RNA melting experiments showed that the decreased ATPase activity of rho and the slower helicase activity at increased KCl or Mg(OAc)2 concentrations are accompanied by a concomitant increase in the secondary structure of the RNA portion of the helicase substate. In contrast, the ATPase activity of rho in the presence of poly(rC), a synthetic RNA that does not form salt-concentration-dependent secondary structure, was shown to be the same in each of the three monovalent salts. Thus, the salts do not directly affect the structure or conformation of the rho protein or the binding of rho to single-stranded RNA. However, the translocation of rho along RNA was more processive in 150 mM KOAc or KGlu than in 150 mM KCl, while the RNA secondary structure was the same in all three monovalent salts. Therefore, the monovalent salt present in the reaction may directly affect rho-RNA interactions when the RNA substrate can form secondary structure. Helicase experiments with an RNA molecule that does not contain a rho loading-site showed that rho translocates less processively along this potential helicase substrate. These results suggest that the helicase activity of rho may be significantly regulated by RNA secondary structure. In addition, one of the mechanisms to concentrate the activity of rho on transcripts containing unstructured rho loading sites may be that rho translocation along such molecules is more processive than it is along more structured RNA molecules in the cell.
The Escherichia coli rho transcription termination protein is a hexameric helicase, and is believed to function by separating an RNA-DNA hybrid. Unlike hexameric DNA helicases, where a single strand of DNA passes through the central channel, it has been proposed that the RNA wraps around the outside of the ring. We have generated a three-dimensional reconstruction of rho, and localized a tRNA molecule bound to the primary RNA-binding site to the outside of the ring. An atomic structure of the N-terminal domain of rho fits into our reconstruction uniquely, with the residues involved in RNA-binding on the outside of the ring. Although rho shares a common structural core with the F1-ATPase and other hexameric helicases, there has been a divergence in function due to rho's N-terminal domain, which has no homology to other helicases.
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Cryoelectron microscopy has been used to visualize the Escherichia coli transcription termination protein rho in a vitreously frozen state, without the use of strains, fixatives or other chemical perturbants. In the absence of RNA cofactor, a variety of structures are observed, reflecting the heterogeneity of complexes formed by rho at protein concentrations near the physiological range (3 to 10 microM). One of the most common structural motifs we see is a six-membered ring of rho subunits (present as either a closed or "notched" circle), which corresponds to the predominant hexameric association state of the protein. Also visible are smaller oligomeric structures, present as curved lines of rho subunits, which probably represent the lower association states of the protein that coexist with the hexamer at these protein concentrations. Addition of oligomers of ribocytosine (rC) of defined lengths (23-mers and 100-mers) results in the generation of more homogeneous populations of rho oligomers. In the presence of (rC)23, all identifiable particles appear either as closed or as notched hexameric circles. A small fraction of these particles are of visibly higher density, and are identified with the dodecamers expected as a subpopulation of rho under these conditions. Binding of (rC)100, an oligomer of length greater than that needed to span the entire hexamer binding site, results in a uniform population of closed circular hexamers. In some images additional features are visible at either the centers or the peripheries of the particles. These features may correspond to the excess length of the rC strands bound to the hexamers. The distributions of particles observed under the various experimental conditions used correlate well to those deduced from physical biochemical studies Seifried et al., accompanying paper).
A plasmid system has been constructed which allows high-level expression of the rho gene of Escherichia coli under the control of the pL promoter and the N-antitermination regulatory system of bacteriophage lambda. The pL-directed synthesis of Rho crucially depends on the lambda N gene product and is promoted most effectively when this product is supplied from the N gene cloned on a separate compatible plasmid with a moderate copy number. The requirement for N can be circumvented partly, but not completely, by deletion of the region preceding the rho structural gene. Attempts were also made to optimize the construction of rho-expression plasmids by adjusting the orientation and location of pL and rho inserts on the pBR322 vector. With optimal conditions, Rho protein is overexpressed 100-fold and can become as much as 10% of the total cellular protein. Using this plasmid system, Rho can be purified with a yield of more than 20 mg from 10 g of induced cells.
Rho is a ring-shaped hexameric motor protein that translocates along nascent mRNA transcript and terminates transcription of select genes in bacteria. Using a numerical optimization algorithm that simultaneously fits all of the presteady-state ATPase kinetic data, we determine how Rho utilizes the chemical energy of ATP hydrolysis to translocate RNA. A random hydrolysis mechanism is ruled out by the observed inhibition of ATPase in a mixed hexamer containing wt and an inactive Rho mutant. We propose a mechanism in which (1) all six subunits are catalytically competent and hydrolyze ATP sequentially, (2) translocation of RNA is driven by the weak to tight binding transition of nucleotide in the catalytic site, (3) hydrolysis is coordinated between adjacent subunits by the transmission of stress via the catalytic arginine finger, (4) hydrolysis weakens the affinity of a subunit for RNA, and (5) the slow release of inorganic phosphate is controlled by changes in circumferential stress around the ring.