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PubMed1984

Binding of rho factor to Escherichia coli RNA polymerase mediated by nusA protein.

The E. coli transcription termination factor rho binds specifically to purified nusA protein. Since nusA protein binds tightly to RNA polymerase, this provides a way of coupling rho to the elongating RNA polymerase complex through protein-protein interactions. These rho-nusA interactions may play a role in modulating rho action at certain terminators and could also be important in the action of antitermination factors such as lambda N protein.

Adenosine Triphosphatases↗
PubMed1976

Isolation and characterization of conditional lethal mutants of Escherichia coli defective in transcription termination factor rho.

Polarity suppressor mutants that are conditional lethal for growth have been isolated in E. coli K12. The mutations map between the ilv and cya loci of the E. coli chromosome. Rho factor isolated from one of these ts mutants does not show transcription termination activity at any temperature tested; however, it is found to be temperature sensitive for its poly(C)-dependent ATPase activity. Unlike the previously known polarity suppressor mutants (suA and psu), the rho mutation suppresses all types of polarity. Other interesting properties of these mutants include ultraviolet sensitivity, recombination deficiency, and decreased ability to lysogenize temperate phages lambda and P1. Our results suggest that rho has an essential function in the growth and normal physiology of cells. The rho(ts) mutant allows the growth of phage lambda defective in the N gene. This result supports the model that N gene product prevents transcription termination by antagonizing rho activity.

Adenosine Triphosphatases↗
PubMed1999

Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis.

The expression and activity of transcription termination factor Rho and the requirement for transcription elongation factors NusA and NusG was investigated in Bacillus subtilis. Rho was present at < 5% of the level found in Escherichia coli, but Rho factors from these two bacteria had similar properties as RNA-activated ATPases and in vitro termination of transcription on the lambda tR1 terminator. The B. subtilis rho gene was autoregulated at the level of transcription; autoregulation required sequences within the rho mRNA leader region and gene. To date, the B. subtilis rho is the only gene from a Gram-positive bacterium found to be regulated by Rho. Rho was not involved in bulk mRNA decay in B. subtilis. The E. coli elongation factors NusA and NusG target Rho, and the importance of these proteins in B. subtilis was examined by gene disruption. The B. subtilis NusG was inessential for both the viability and the autoregulation of Rho, whereas NusA was essential, and the requirement for NusA was independent of Rho. This contrasts with E. coli in which NusG is essential but NusA becomes dispensable if Rho terminates transcription less efficiently.

Adenosine Triphosphate↗
PubMed1980

Transcription-termination factor Rho from Bacills subtilis.

A protein has been isolated from Bacillus subtilis which has functions similar to that of transcription termination factor rho (rho) from Escherichia coli. The apparent molecular weight of the B. subtilis rho factor is about 80000-95000 as estimated by a non-denaturing polyacrylamide gel electrophoresis method. It contains two subunits with a molecular weight of 47000 as determined by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The rho factor shows poly(C)-dependent beta-gamma ATPase activity and depresses the activity of RNA synthesis from B. subtilis phage rho 29 DNA template with purified B. subtilis RNA polymerase holoenzyme. The specific activity of the poly(C)-dependent ATPase of the B. subtilis rho factor was significantly less than that of the E. coli rho factor. In the presence of rho factor fewer RNA transcripts were produced overall from the rho 29 template and smaller RNA transcripts with discrete sizes were made. These results suggest that the B. subtilis rho factor can catalyze transcription termination at specific sites on rho 29 phage DNA in vitro.

Adenosine Triphosphatases↗
PubMed1998

Crystal structure of the RNA-binding domain from transcription termination factor rho.

Transcription termination factor rho is an ATP-dependent hexameric helicase found in most eubacterial species. The Escherichia coli rho monomer consists of two domains, an RNA-binding domain (residues 1-130) and an ATPase domain (residues 131-419). The ATPase domain is homologous to the beta subunit of F1-ATPase. Here, we report that the crystal structure of the RNA-binding domain of rho (rho130) at 1.55 A confirms that rho130 contains the oligosaccharide/oligonucleotide-binding (OB) fold, a five stranded beta-barrel. The beta-barrel of rho130 is also surprisingly similar to the N-terminal beta-barrel of F1 ATPase, extending the applicability of F1 ATPase as a structural model for hexameric rho.

Crystallography, X-Ray↗
PubMed1980

Studies of RNA release reaction catalyzed by E. coli transcription termination factor rho using isolated ternary transcription complexes.

Protein factor rho catalyzes site-specific termination of transcription in a reaction requiring hydrolysis of nucleoside triphosphate with eventual release of RNA from RNA polymerase and DNA template. We have characterized the rho-catalyzed RNA release reaction using isolated transcription complexes. Transcription complexes containing T7 D111 DNA, RNA polymerase, and 3H-labeled nascent RNA were formed and isolated by gel filtration on an Agarose 5M column. When the ternary complexes were incubated with rho factor in the presence of ATP, or dATP, significant amounts of nascet RNA were released from the complexes as determined in a membrane filtration assay. Gel electrophoretic analysis of RNA has revealed that rho releases selected species of discrete-sized RNA from among those originally present in the ternary complexes. These results show that rho essentially acts to release RNA from those ternary complexes which have come to pause, and that this reaction proceeds in a discrete step separately from the pausing of RNA synthesis. Under the conditions used, the extent of RNA release widely varied at individual pausing sites and thus the action of rho exhibited certain site-selectivity.

Adenosine Triphosphate↗
PubMed1999

The mechanism of ATP hydrolysis at the noncatalytic sites of the transcription termination factor Rho.

Escherichia coli transcription termination factor rho is a hexamer with three catalytic subunits that turnover ATP at a fast rate and three noncatalytic subunits that turnover ATP at a relatively slow rate. The mechanism of the ATPase reaction at the noncatalytic sites was determined and was compared with the ATPase mechanism at the catalytic sites. A sequential mechanism for ATP binding or hydrolysis that was proposed for the catalytic sites was not observed at the noncatalytic sites. Pre-steady-state pulse-chase experiments showed that three ATPs were tightly bound to the noncatalytic sites and these were simultaneously hydrolyzed at a rate of 1.8 s(-1) at 18 degrees C. The apparent bimolecular rate constant for ATP binding was determined as 5.4 x 10(5) M(-1) s(-1) in the presence of poly(C) RNA. The ATP hydrolysis products dissociated from the noncatalytic sites at 0.02 s(-1). The hydrolysis of ATP at the noncatalytic sites was at least 130 times slower, and the overall ATPase turnover was 1500 times slower than that at the catalytic sites. These results from studies of the rho protein are likely to be general to hexameric helicases. We propose that the ATPase activity at the noncatalytic site is too slow to drive translocation of the protein on the nucleic acid or to provide energy for nucleic acid unwinding.

Adenosine Triphosphatases↗
PubMed2005

Active site occupancy required for catalytic cooperativity by Escherichia coli transcription termination factor Rho.

Escherichia coli transcription termination factor Rho exhibits the phenomenon of catalytic cooperativity. The catalytic rate per site is 30-fold faster when all three sites are filled with substrate ATP than when only a single site is occupied (Stitt, B. L., and Xu, Y. (1998) J. Biol. Chem. 273, 26477-26486). Experiments presented here investigate whether all three active sites must be filled or whether only two occupied sites are required for catalytic cooperativity. The results indicate that all three Rho catalytic sites must be filled with substrate to achieve the enhanced catalytic rate, both in pre-steady-state and in steady-state hydrolysis. They further suggest that, once the enzyme is saturated with ATP, a V(max) enzyme conformation is achieved that is stable for at least three catalytic cycles.

Adenosine Triphosphate↗
PubMed2002

Analysis of tryptophanase operon expression in vitro: accumulation of TnaC-peptidyl-tRNA in a release factor 2-depleted S-30 extract prevents Rho factor action, simulating induction.

Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. The key feature of this antitermination mechanism has been shown to be the retention of uncleaved TnaC-peptidyl-tRNA in the translating ribosome. This ribosome remains stalled at the tna stop codon and blocks the access of Rho factor to the tna transcript, thereby preventing transcription termination. In normal S-30 preparations, synthesis of a TnaC peptide containing arginine instead of tryptophan at position 12 (Arg(12)-TnaC) was shown to be insensitive to added tryptophan, i.e. Arg(12)-TnaC-peptidyl-tRNA was cleaved, and there was normal Rho-dependent transcription termination. When the S-30 extract used was depleted of release factor 2, Arg(12)-TnaC-tRNA(Pro) was accumulated in the absence or presence of added tryptophan. Under these conditions the accumulation of Arg(12)-TnaC-tRNA(Pro) prevented Rho-dependent transcription termination, mimicking normal induction. Using a minimal in vitro transcription system consisting of a tna template, RNA polymerase, and Rho, it was shown that RNA sequences immediately adjacent to the tnaC stop codon, the presumed boxA and rut sites, contributed most significantly to Rho-dependent termination. The tna boxA-like sequence appeared to serve as a segment of the Rho "entry" site, despite its likeness to the boxA element.

Arginine↗
PubMed1982

rho Factors from polarity suppressor mutants with defects in their RNA interactions.

rho proteins isolated from strains of Escherichia coli with the suA1, suA100, and suA120 polarity suppressor mutant alleles of the rho gene are shown to be defective in termination of T7 DNA transcription in vitro. These mutant rho factors are found to be less active than normal rho in catalyzing the hydrolysis of ATP with isolated T7 RNA as cofactor, and their lower ATPase activities correlate well with their decreased abilities to mediate release of RNA from isolated T7 DNA transcription complexes. The mutant rho factors also have lower ATPase activities than wild type rho with certain synthetic RNA polymers, but they have full activity with poly(C). Because the mutant rho factors have normal ATPase activity with some RNAs, we conclude that their defects are in their abilities to interact with certain RNA cofactors and not in their catalytic capacities. Binding studies indicate that their defects do not appear to involve the primary interaction between rho and RNA. When poly(dC) is used to saturate the primary site for RNA on rho, the Km values for (Cp)7C as a secondary site activator of rho ATPase are 3.4, 80, and 15 microM for rho from the strains with the wild type, suA1, and suA100 alleles, respectively. These results suggest that the defects in the rho factors isolated from strains with the polarity suppressor alleles suA1 and suA100 involve their abilities to interact with RNA at their secondary sites.

Adenosine Triphosphatases↗
PubMed1995

Structural and functional dissections of transcription termination factor rho by random mutagenesis.

Transcription termination factor rho from Escherichia coli is a homohexamer of 419 amino acid subunits and catalyzes an ATP-dependent release of nascent RNA transcripts. A rho monomer has three distinct domains functioning independently at the first approximation: the amino-terminal one quarter containing a primary RNA-binding site, the central 270-amino acids region constituting an ATP-binding domain with homologies to F1-ATPase, and the carboxy-terminal remainder with unknown function(s). To further delineate the structural and functional organizations of rho protein, we undertook its random mutagenesis using error-prone polymerase chain reactions with the carboxy-terminal 100-amino acid region chosen as the initial target. From 14 mutants identified, rho protein was purified and characterized in vitro. Of these, 11 mutants are defective in termination in vivo and show decreased activities in various partial functions examined: ATP binding; RNA binding; and ATPase activities dependent on three cofactors with decreasing efficacies, poly(C), lambda cro RNA and poly(U). A few of them are also affected in the putative secondary RNA-binding site that is functionally coupled to ATP hydrolysis. By contrast, the three other mutants are hyperactive in termination, poly(U)-dependent ATPase activity, and RNA interaction at the primary site. In these properties, the hyper-terminating mutants strikingly resemble the "super rho" mutant formerly found in the amino-terminal domain. Taken together, these findings indicate that the carboxy-terminal region plays a pivotal role in functionally coupling the RNA and ATP-binding domains, plausibly by acting as an interface for their interaction within or across individual subunits. In light of the reported X-ray crystallographic structure of F1-ATPase, we propose a model for the tertiary and quaternary structure of rho that is consistent with the observed mutational effects as well as a number of structural and functional properties characteristic of rho.

Adenosine Triphosphate↗
PubMed1996

Mutational analysis and secondary structure model of the RNP1-like sequence motif of transcription termination factor Rho.

The function of transcription termination factor Rho from Escherichia coli is dependent upon its ability to bind to specific sites on nascent RNA molecules. The roles of 19 individual amino acid residues (Ile49 to Ser67) in and near a phylogenetically conserved sequence segment of Rho that is similar to the RNP1 motif found in many RNA-binding proteins were examined by testing the phenotypic consequences of mutational changes that were introduced into rho by a random-sequence cassette mutagenesis procedure. The tests of each mutant included the ability of the cells to survive at 42 degrees C in the absence of wild-type rho, the efficiency of termination at a Rho-dependent terminator (lambdatR1) in vivo, the relative level of expression of the mutant protein, and the ability of some of the mutant proteins to bind RNA. The results revealed that residues in the RNP1-like sequence of DGFGFLR (residues 60 to 66) were more important than residues 49 to 59 for termination function and RNA binding, and identified three residues that were particularly sensitive to mutation: Asp60, Phe62 and Arg66. The properties of the mutants are consistent with a secondary structure model, derived from phylogenetic analysis, that has the RNP1-like sequence on one of the three beta-strands of an antiparallel beta-sheet with Asp60 and Gly61 in a turn and the side-chains of Phe62, Phe64 and Arg66 accessible on the same face of the beta-structure for interaction with RNA.

Amino Acid Sequence↗
PubMed1997

Function of the novel subdomain in the RNA binding domain of transcription termination factor Rho from Micrococcus luteus.

Transcription termination factor Rho from Micrococcus luteus, a high G + C Gram-positive bacterium, contains an unusual extra sequence within its RNA binding domain that is rich in Arg, Glu, and Asp residues and deficient in hydrophobic residues. To determine the role of this extra sequence, we compared the biochemical properties of a variant lacking nearly all the extra sequence, des(60-300) Rho, to that of wild-type M. luteus Rho. The two forms had very similar properties except that the des(60-300) Rho was unable to terminate transcription with Escherichia coli RNA polymerase at the promoter proximal sites used by the wild-type Rho on a lambda cro DNA template but could cause termination at more distal sites and did cause termination at proximal sites when ITP replaced GTP in the reaction mixture. The RNA binding properties of the two forms of this Rho with normal and inosine-substituted RNAs were found to correlate fully with their termination properties. These results indicate that the arginine-rich extra sequence is directly involved in the selection of the termination site and support the hypothesis that the sequence is present in M. luteus Rho to facilitate its binding to M. luteus transcripts, which are likely to have a high degree of base-paired secondary structure because of their high proportion of G residues.

Adenosine Triphosphatases↗
PubMed2001

The kinetic pathway of RNA binding to the Escherichia coli transcription termination factor Rho.

The Escherichia coli transcription termination factor Rho is structurally and functionally homologous to hexameric helicases that assemble into ring structures. Using stopped-flow fluorescence and presteady-state ATPase kinetics, we have determined the kinetic pathway of poly(C) RNA binding to Rho hexamer, both in the presence and in absence of ATP. These studies indicate a four-step sequential mechanism of RNA binding and reveal the respective roles of the primary and secondary RNA binding sites in initiation and ATPase activation of Rho. The primary RNA binding sites of Rho hexamer interact with poly(C) RNA at a diffusion-limited rate constant close to 8 x 10(8) m(-1) s(-1), resulting in the Rho-RNA species PR1, which subsequently isomerizes to PR2 with a rate constant 21 s(-1). The PR2 isomerizes to PR3 with a rate constant of 32 s(-1) in the presence of ATP, and the formation of PR4 from PR3 results in a species that is fully competent in hydrolyzing ATP at the RNA-stimulated rate. The PR3 to PR4 isomerization occurs at a relatively slow rate of 4.1 s(-1); thus, the presteady-state ATPase kinetics show a distinct lag due to the slow initiation step. The interactions of the RNA with the primary sites trigger ring opening, and we propose that during the last two steps, the RNA migrates into the central channel and interacts with the secondary sites, resulting in the activation of the ATPase activity. The primary RNA binding sites, in addition to promoting sequence specific initiation, kinetically facilitate loading of the RNA into the secondary sites, which are relatively inaccessible, since they are present in the central channel. These studies reveal common features used by hexameric helicases to bind nucleic acids in an efficient and specific manner.

Adenosine Triphosphatases↗
PubMed1997

Transcription termination factor Rho is essential for Micrococcus luteus.

The growth of Micrococcus luteus, a soil microorganism that belongs to the high-G+C gram-positive phylogenetic group, is prevented by bicyclomycin, an antibiotic that inhibits the activity of the M. luteus transcription termination factor Rho. A mutant that can grow in 0.3 mM bicyclomycin has a Rho that is insensitive to bicyclomycin and has the single amino acid residue change of Asp474 to Gly. These results indicate that the function of its Rho factor is essential for M. luteus and that growth of a gram-positive organism can be blocked by bicyclomycin.

Anti-Bacterial Agents↗
PubMed2002

The transcription termination factor Rho is required for oxidative stress survival in Caulobacter crescentus.

A transposon Tn5 mutagenesis library was generated from Caulobacter crescentus strain NA1000, and clones with deficiency in survival in a high concentration of NaCl were selected. One of these clones, 37G10, has the Tn5 integrated within the coding region of the transcription termination factor Rho. Analysis of this mutant phenotype showed that the cells are motile and present a normal cell cycle, but have a longer generation time. This strain is sensitive to acidic pH, to the presence of different salts and to heat shock, but it responds well to UV light and alkaline pH. The most striking phenotype of the rho mutant is that it is extremely sensitive to oxidative stress, in both exponential and stationary phases. Experiments using a transcriptional fusion of the rho promoter region to the lacZ gene showed that rho gene expression varies during the cell cycle, showing very low expression levels at the swarmer cell stage and presenting maximum levels in early predivisional cells. Transcription of the rho gene is increased in the rho mutant strain, which is indicative of an autoregulatory circuit, and there is a small variation in the cell cycle pattern of expression. Several peptides have their synthesis altered in the mutant strain, as analysed by two-dimensional gel electrophoresis, most of which show a reduction in expression. These results indicate that the Rho factor is essential for an efficient response to certain stresses in Caulobacter.

Amino Acid Sequence↗
PubMed1992

ATPase activity of transcription-termination factor rho: functional dimer model.

Transcription-termination factor rho of Escherichia coli functions as an RNA-dependent ATPase that causes transcript release at specific rho-dependent termination sites on the DNA template. Rho exists as a hexagon of identical subunits, physically organized as a trimer of dimers with D3 symmetry. The structural asymmetry of the dimer is reflected in the binding properties of rho; each dimer has a strong and a weak binding site for both the ATP substrate and the RNA cofactor. Here we use homopolynucleotides in competition and complementation experiments to characterize the ATPase activation properties of the cofactor binding sites of the functional rho dimer. We show that (i) no ATPase activity is observed unless both the high- and the low-affinity cofactor binding sites of the functional rho dimer are occupied; (ii) saturating levels of poly(rC), poly(rC) in combination with poly(rU), or poly(rU) alone can fully activate the ATPase of rho; and (iii) poly(dC) can serve as a fully competitive inhibitor of half of the ATPase activity of rho when one of the cofactor sites is filled with poly(rC). These observations lead to a set of phenomenological rules that describe the cofactor dependence of the ATPase activation of the functional dimer of rho and help to define a mechanistic basis for interpreting rho function in termination.

Adenosine Triphosphatases↗