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• W2109503950 abstract "Model studies have identified 16 conserved positively charged amino acids that form a positive strip pointing toward the center hole of Rho. Fourteen residues were individually changed to either an alanine or a glycine and one to a glutamate. Residues Arg269, Arg272, Lys283, Arg296, Lys298, and Arg299 form a subdomain (locus) located N-terminal to (above) the ATP hydrolysis domain (P-loop) and mutations in these residues led to either inactive Rho or to proteins displaying decreasedkcat for poly(C)-dependent ATP hydrolysis, increased Km for ribo(C)10 activation, and decreased transcription termination efficiencies (57–77%) compared with wild-type Rho. Residues Arg347, Lys348, Lys352, and Arg353 form a subdomain (locus) C-terminal to (below) the ATP hydrolysis domain, and mutations in these residues also show a decreased kcat for poly(C)-dependent ATP hydrolysis, an increasedKm for ribo(C)10 activation, and a 50–70% decrease in transcription termination, compared with wild-type Rho. Residues Arg212 and Lys336 surround the ATP hydrolysis domain, and mutations in these residues also altered the kinetic properties of Rho. We conclude that the secondary RNA-tracking site consists of amino acids whose putative orientation faces the central hole in Rho and in part reside in two clusters of positively charged residues located above and below the ATP hydrolysis domain. Model studies have identified 16 conserved positively charged amino acids that form a positive strip pointing toward the center hole of Rho. Fourteen residues were individually changed to either an alanine or a glycine and one to a glutamate. Residues Arg269, Arg272, Lys283, Arg296, Lys298, and Arg299 form a subdomain (locus) located N-terminal to (above) the ATP hydrolysis domain (P-loop) and mutations in these residues led to either inactive Rho or to proteins displaying decreasedkcat for poly(C)-dependent ATP hydrolysis, increased Km for ribo(C)10 activation, and decreased transcription termination efficiencies (57–77%) compared with wild-type Rho. Residues Arg347, Lys348, Lys352, and Arg353 form a subdomain (locus) C-terminal to (below) the ATP hydrolysis domain, and mutations in these residues also show a decreased kcat for poly(C)-dependent ATP hydrolysis, an increasedKm for ribo(C)10 activation, and a 50–70% decrease in transcription termination, compared with wild-type Rho. Residues Arg212 and Lys336 surround the ATP hydrolysis domain, and mutations in these residues also altered the kinetic properties of Rho. We conclude that the secondary RNA-tracking site consists of amino acids whose putative orientation faces the central hole in Rho and in part reside in two clusters of positively charged residues located above and below the ATP hydrolysis domain. Rho transcription termination factor is one of several nucleic acid-binding proteins belonging to a family of helicases with a homohexameric structure shaped like a toroid ring that utilizes the hydrolysis of ATP to move along the nucleic acid (1Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (665) Google Scholar, 2Bird L.E. Subramanya H.H. Wigley D.B. Curr. Opin. Struct. Biol. 1998; 8: 14-18Crossref PubMed Scopus (147) Google Scholar). Rho protein binds to a poorly defined cytosine-rich sequence on nascent RNA called a rut site (Rho utilization), which contains little or no secondary structure. Rut sites are usually found upstream from the start of specific genes or between genes in operons (3Richardson J.P. J. Biol. Chem. 1982; 257: 5760-5766Abstract Full Text PDF PubMed Google Scholar), and once Rho binds to the rut site, it tracks 5′ to 3′ toward the stalled RNA polymerase. In a poorly defined mechanism that may require helicase activity (4Brennan C.A. Dombroski A.J. Platt T. Cell. 1987; 48: 945-952Abstract Full Text PDF PubMed Scopus (181) Google Scholar), Rho disrupts the polymerase-transcript complex, thereby terminating transcription. Bicyclomycin, a commercial antibiotic, has been shown to inhibit Rho function (5Zwiefka A. Kohn H. Widger W.R. Biochemistry. 1993; 32: 3564-3570Crossref PubMed Scopus (89) Google Scholar) specifically by interfering with the tracking of Rho (6Magyar A. Zhang X. Kohn H. Widger W.R. J. Biol. Chem. 1996; 271: 25369-25374Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Bicyclomycin inhibits the growth of many Gram-negative organisms, including Escherichia coli, Shigella, and Salmonella (7Williams R.M. Durham C.A. Chem. Rev. 1988; 88: 511-540Crossref Scopus (64) Google Scholar) and at least one Gram-positive bacterium, Micrococcus luteus (8Nowatzke W.L. Keller E. Koch G. Richardson J.P. J. Bacteriol. 1997; 179: 5238-5240Crossref PubMed Google Scholar), inferring the vital nature of Rho for cellular function (9Das A. Court D. Adhya S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 1959-1963Crossref PubMed Scopus (146) Google Scholar, 10Inoko H. Shigesada K. Imai M. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 1163-1166Crossref Scopus (32) Google Scholar, 11Richardson J.P. Carey III, J.L. J. Biol. Chem. 1982; 257: 5767-5771Abstract Full Text PDF PubMed Google Scholar). The Rho monomer consists of 419 amino acid residues divided into two structural domains, a primary RNA-binding domain (residues 1–151) and the ATP hydrolysis domain (residues 167–419), which is based upon structural similarity with F1-ATP synthase (12Dombroski A.J. LaDine J.R. Cross R.L. Platt T. J. Biol. Chem. 1988; 263: 18810-18815Abstract Full Text PDF PubMed Google Scholar). The RNA-binding domain contains a DGFGFLR (amino acid residues 60–66) conserved RPN-1 RNA recognition motif (13Martinez A. Burns C.M. Richardson J.P. J. Mol. Biol. 1996; 257: 909-918Crossref PubMed Scopus (32) Google Scholar, 14Brennan C.A. Platt T. J. Biol. Chem. 1991; 266: 17296-17305Abstract Full Text PDF PubMed Google Scholar). A solution structure of the N-terminal 130 residues of Rho has been solved using NMR techniques (15Briercheck D.M. Wood T.C. Allison T.J. Richardson J.P. Rule G.S. Nat. Struct. Biol. 1998; 5: 393-399Crossref PubMed Scopus (60) Google Scholar) and a crystal structure is also available (16Allison T.J. Wood T.C. Briercheck D.M. Rastinejad F. Richardson J.P. Rule G.S. Nat. Struct. Biol. 1998; 5: 352-356Crossref PubMed Scopus (67) Google Scholar). The primary RNA-binding domain sits as a cap on the toroid ring, distinct from the ATP hydrolysis domain, and binds RNA tightly. ATPase activity is induced by the addition of poly(C) to Rho. Substitution of poly(dC) for poly(C) leads to tight Rho binding but no ATP hydrolysis. The addition of short oligoribonucleotides (7–10 residues long with a predominance of cytosine residues) to the Rho-poly(dC) complex activates ATPase activity. The activation by ribo(C)10 is thought to occur at the secondary RNA-binding/tracking site (3Richardson J.P. J. Biol. Chem. 1982; 257: 5760-5766Abstract Full Text PDF PubMed Google Scholar). Recently, the projected Q-loop region, positioned on top, inside the toroid ring, facing the central hole of Rho, has been implicated in RNA binding, and this RNA-binding domain changes conformation upon binding ATP (17Wei R.R. Richardson J.P. J. Biol. Chem. 2001; 276: 28380-28387Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). A model for Rho tracking coupled to ATP hydrolysis that relies on the structural similarity between Rho and the β-subunit of F1-ATP synthase and utilizes residues facing inside of the central hole in Rho has been put forth (18Vincent F. Openshaw M. Trautwein M. Gaskell S.J. Kohn H. Widger W.R. Biochemistry. 2000; 39: 9077-9083Crossref PubMed Scopus (19) Google Scholar). A strip of positively charged amino acids on Rho positioned toward the inside of the hole was identified based on the structure of bovine F1-ATP synthase (12Dombroski A.J. LaDine J.R. Cross R.L. Platt T. J. Biol. Chem. 1988; 263: 18810-18815Abstract Full Text PDF PubMed Google Scholar, 19Magyar A. Zhang X. Abdi F. Kohn H. Widger W.R. J. Biol. Chem. 1999; 274: 7316-7324Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) threaded with E. coli Rho sequence and energy minimized (18Vincent F. Openshaw M. Trautwein M. Gaskell S.J. Kohn H. Widger W.R. Biochemistry. 2000; 39: 9077-9083Crossref PubMed Scopus (19) Google Scholar). This model takes into account three active ATP hydrolysis sites per hexamer, sequential hydrolysis of ATP, Rho activation by short oligoribonucleotides, and the discrimination between hydrolytic and inactive subunits. The strip of positive charges was projected to bind the polyphosphate backbone of the RNA as Rho translocates toward the RNA polymerase. The positive charges were clustered into two separate loci found near the N and C termini surrounding the ATP hydrolysis domain. In this paper, we report the effects of site mutations carried out on these amino acid residues. We document the importance of positive charges for Rho to hydrolyze ATP, to bind RNA, and to terminate transcripts. Bicyclomycin was kindly provided by Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan) and was further purified by three successive silica gel chromatographies using 20% methanol-chloroform as the eluant (20Park H. Zhang X. Moon H. Zwiefka A. Cox K. Gaskell S.J. Widger W.R. Kohn H. Arch. Biochem. Biophys. 1995; 323: 447-454Crossref PubMed Scopus (39) Google Scholar). Oligonucleotide primers were synthesized by Genosys Biotechnology, Inc. (The Woodlands, TX). T4 polynucleotide kinase, T4 DNA ligase, and restriction enzymes were purchased from Promega Co. (Madison, WI). Pfu DNA polymerase was obtained from Stratagene (La Jolla, CA). The metal chelating column was from Amersham Biosciences. Radionucleotides [γ-32P]ATP and [α-32P]CTP (3000 Ci/mmol) were purchased from PerkinElmer Life Sciences, and nucleotides and RNase inhibitors were from Ambion, Inc. (Austin, TX). Polyethylenimine thin-layer chromatography plates used for ATPase assays were purchased from J. T. Baker, Inc. Ribo(C)10 was obtained from Oligos Etc. (Wilsonville, OR). All other chemicals were reagent grade. The plasmid pET-RhoW that contained wild-type rho (19Magyar A. Zhang X. Abdi F. Kohn H. Widger W.R. J. Biol. Chem. 1999; 274: 7316-7324Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) was used as a template for DNA amplification. Overlapping primers with the desired mutation were used to introduce base changes, as described in the QuikChangeTM site-directed mutagenesis kit (Stratagene). The resulting PCR-amplified plasmid DNA was digested withDpnI and transformed into host strain JM109. Isolated plasmid DNA from the transformed cells was sequenced with an Applied Biosystems 377 sequencer using the Big Dye reaction kit to identify sequences with specific site changes. The entire rho gene was sequenced to ensure that no other mutations were present in the singly mutated gene. One of four expression systems were used to generate soluble and functional mutant Rho proteins: the pET14b expression vector using host strain BL21(DE3)pLysS (T7 polymerase) (Novagen), the pET14b vector in the salt-induced T7 polymerase host BL21SI (Invitrogen), the pBAD33 vector (arabinose induction) using host strain MG1655 (21Guzman L.-M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3876) Google Scholar), and the pET14b vector with the λ host HMS174 expressed by introducing T7 polymerase by transfection with λ phage CE6 (Novagen). For expression using host BL21(DE3)pLysS, HSM174, or BL21SI, the original pET14b vector was used. Expression of mutant Rho using the arabinose promoter in the vector pBAD33 required excising the XbaI-HindIII fragment of the pET14b vector that contained the entire rhogene including the upstream SD sequence but shy of the T7 promoter, and ligating it into a similarly digested pBAD33 vector. Recombinant plasmids were transformed into host MG1655, and expression was induced by adding 0.2% arabinose to cells. To isolate Rho, cells were centrifuged, resuspended, and lysed by the addition of lysozyme (130 μg ml−1) and 0.05% deoxycholate. After brief sonication using a Branson sonicator, the cell debris was removed by centrifugation and the resulting supernatant was placed on a metal chelating column (AmershamBiosciences) bound with Ni2+. After washing, the bound protein Rho was recovered using a linear gradient of 0–0.5m imidazole. SDS electrophoresis of the isolated Rho showed only one peptide at 47,000 Da. Rho protein was dialyzed against storage buffer and kept at −80 °C until used. Poly(C)-dependent ATP hydrolysis activity was measured as described previously (19Magyar A. Zhang X. Abdi F. Kohn H. Widger W.R. J. Biol. Chem. 1999; 274: 7316-7324Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) varying the ATP concentrations from 9.1 to 100 μm. The reactions were initiated by adding 0.5 μCi of [γ-32P]ATP and varying concentrations of ATP to a solution of 40 mm Tris-HCl, pH 7.9, 50 mm KCl, 12 mm MgCl2, 0.1 mm EDTA, 0.1 mm dithiothreitol, 1.4 mg ml−1 bovine serum albumin, 40 nm poly(C), and Rho (33 nmol of monomer). The assays were carried out as described (6Magyar A. Zhang X. Kohn H. Widger W.R. J. Biol. Chem. 1996; 271: 25369-25374Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) using 200 nm Rho protein monomer. The two different ranges of ribo(C)10 concentrations used depended upon theKm (ribo(C)10) values. A range ribo(C)10 between 0.7 and 20 μm was used for wild-type Rho, whereas a range of ribo(C)10 between 20 and 200 μm was used when the Km indicated a value much greater than the wild-type. Estimates ofKm were reported for values that exceeded 500 μm because of the limited ribo(C)10concentrations that could be used. Transcription termination assays were carried out using a modified trp operon (22Wu A.M. Christie G.E. Platt T. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2913-2917Crossref PubMed Scopus (81) Google Scholar), as described (6Magyar A. Zhang X. Kohn H. Widger W.R. J. Biol. Chem. 1996; 271: 25369-25374Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) with slight modifications. In brief, the reaction was carried out in a 10-μl volume with 200 μm ATP, UTP, and GTP and 20 μm CTP, 7 μCi of [α-32P]CTP, 0.1 pmol of template, 0.4 units/μl RNase inhibitor, 0.01 μg/μlE. coli RNA polymerase, and 70 nm Rho protein. Transcription termination was measured in the absence and presence of mutant Rho protein and in the presence of Rho plus 50 μmbicyclomycin. The transcripts were separated and visualized as described (6Magyar A. Zhang X. Kohn H. Widger W.R. J. Biol. Chem. 1996; 271: 25369-25374Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Subtracting the digitized background from both the read through and the Rho-terminated transcripts allowed quantitation of the termination reactions. The background from the wild-type Rho was subtracted from all the mutants and the percentage of termination was calculated as the amount of terminated transcripts divided by the amount of read through transcript plus the amount of terminated transcript times 100. Trp t′ RNA was made using theSalI linearized pTRP5 plasmid and T7 RNA polymerase. The reaction was carried out in 100 μl of transcription buffer (40 mm Tris-HCl, 15 mm MgCl2, 5 mm dithiothreitol, and 0.5 mg/ml acetylated bovine serum albumin), 1 mm each of ATP, GTP, and UTP, and 24 μm CTP, 250 μCi of [α-32P]CTP, 40 units of RNase inhibitor, and 25 μg of DNA. The reaction was started with 1.4 μg of T7 RNA polymerase. The reaction was incubated at 42 °C for 2 h and another 0.56 μg of enzyme was added and incubated for another 2 h. RNase-free DNase (1 μl) was added and the RNA was purified using a RNeasyTM kit (Qiagen). Purity was determined by urea-PAGE and the RNA concentration was determined byA260. Binding of trp t′ RNA was carried out in binding buffer (40 mm Tris-HCl, pH 8.0, 25 mm KCl, 10 mm MgCl2, 0.1 mm dithiothreitol, and 0.1 mm EDTA). Binding reaction was started by adding labeled trp t′ RNA to a final concentration of 1.2 nm to Rho varying from 0 to 12 nm (hexamer) and incubating at 25 °C for 10 min, followed by filtration of the binding reaction through an S&S nitrocellulose filter presoaked in 1× binding buffer with 0.1 mg/ml denatured yeast RNA. The filter was washed twice with 0.5 ml of binding buffer, dried briefly, and measured with a scintillation counter (6Magyar A. Zhang X. Kohn H. Widger W.R. J. Biol. Chem. 1996; 271: 25369-25374Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The percent of Rho binding RNA was measured at saturating RNA concentrations from the moles of RNA bound to Rho times the binding efficiency of Rho to the filter (24Gan E. Richardson J.P. Biochemistry. 1999; 38: 16882-16888Crossref PubMed Scopus (22) Google Scholar) using published procedures (23Witherall G.W. Uhlenbeck O.C. Biochemistry. 1989; 28: 71-76Crossref PubMed Scopus (90) Google Scholar) and used to estimate the concentration of binding-active Rho. The L50 value, the concentration of Rho that gives half-maximal retention of trp t′ RNA was measured using the binding-active Rho concentrations. The Knd and thenth root of Knd was measured as described (24Gan E. Richardson J.P. Biochemistry. 1999; 38: 16882-16888Crossref PubMed Scopus (22) Google Scholar). The fluorescence of F355W and wild-type Rho was determined using a Cary Eclipse spectrofluorimeter at 100 nm Rho monomer in 40 mm Tris-HCl, pH 7.9, 50 mm KCl, 12 mm MgCl2, 0.1 mm EDTA, and 0.1 mm dithiothreitol. Emission spectra were recorded between 300 and 450 nm by excitation at 280 nm, and the spectra were compared by zeroing at 300 nm. Studies of RNA-protein interactions show that base stacking, geometric complementarity, and coulombic forces contribute to binding (25Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1719) Google Scholar, 26Draper D.E. Annu. Rev. Biochem. 1995; 64: 593-620Crossref PubMed Scopus (198) Google Scholar). A model defining the Rho secondary RNA-binding/RNA-tracking site was proposed based on the symmetry of Rho subunits and the identification of conserved arginine and lysine residues located on the surface of the central hole of the hexameric Rho assembly (18Vincent F. Openshaw M. Trautwein M. Gaskell S.J. Kohn H. Widger W.R. Biochemistry. 2000; 39: 9077-9083Crossref PubMed Scopus (19) Google Scholar). The notion that RNA tracking occurred through the central hole of Rho was based on RNA cross-linking experiments (17Wei R.R. Richardson J.P. J. Biol. Chem. 2001; 276: 28380-28387Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and by homology to known helicases (27Kim D.E. Patel S.S. J. Biol. Chem. 2001; 276: 13902-13910Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). A technique referred to alanine scanning, which converts selected residues to alanine (glycine), was applied to conserved arginine and lysine residues in the central hole of Rho. The loss of positively charged amino acid residues on Rho function was assessed by comparing the kinetic properties of the mutated Rho protein with wild-type. We included an additional Rho mutant, F355W, in our study to further verify the Rho model. Residue Phe355 was homologous to βTyr331 in E. coliF1-ATP synthase. Residue βTyr331 has been implicated in ATP binding (28Weber J. Wilke-Mounts S. Lee R.S.F. Grell E. Senior A.E. J. Biol. Chem. 1993; 27: 20126-20133Abstract Full Text PDF Google Scholar) and was base stacked with the adenosine moiety of ATP (29Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2718) Google Scholar). Accordingly, the replacement of the phenylalanine 355 moiety in Rho with tryptophan provides a potential fluorescence probe sensitive to ATP binding. The plasmid pET-RhoW (19Magyar A. Zhang X. Abdi F. Kohn H. Widger W.R. J. Biol. Chem. 1999; 274: 7316-7324Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), which expresses wild-type Rho with a N-terminal His tag using a pET14b vector with a T7 RNA polymerase system, was used as a template for introducing site mutations. Table I summarizes the residues changed and the effect change had on mutant protein expression in various expression systems. The mutant proteins were first expressed from pET14b in the hosts BL21(DE3)pLysS or BL21SI (salt-induced host). Of the 15 mutants in which positively charged amino acids were replaced, only R212G, R347G, K348A, and wild-type grew in the salt-induced host expression strain. The induction of R347G and K348A with isopropyl-1-thio-β-d-galactopyranoside in BL21(DE3)pLysS caused a pronounced decrease in the growth of the cells. This observation suggested that these specific Rho mutations had a negative effect on cell growth. The remaining mutations, when introduced into the BL21 strains, generated few, if any, transformants and those that grew produced truncated protein although the plasmid was stable in host JM109 (data not shown).Table ISummary of mutations and protein expressionRhoLocusBase changeExpression systemActivityWild-typepET14b-BL21pLysS+R160AC-terminalCGC to GCCpET14b-λCE6-HMS174−R212GN-terminalCGT to GGTpET14b-BL21pLysS+R238AN-terminalCGC to GCCpET14b-λCE6-HMS174+R269AN-terminalCGT to GCTpET14b-λCE6-HMS174−R272AN-terminalCGC to GCCpET14b-λCE6-HMS174−1-aExpression resulted in the formation of insoluble inclusion bodies.K283EN-terminalCGC to GCCpET14b-λCE6-HMS174−R296AN-terminalCGT to GCTpET14b-λCE6-HMS174+K298AN-terminalAAA to GCApBAD33-MG1655+R299AN-terminalCGC to GCCpBAD33-MG1655+K336AN-terminalAAA to GCApBAD33-MG1655+R347GC-terminalCGT to GGTpET14b-BL21pLysS+K348AC-terminalAAG to GCGpET14b-BL21SI+K352AC-terminalAAA to GCApBAD33-MG1655+R353AC-terminalCGC to GCCpET14b-λCE6-HMS174+R384AC-terminalCGC to GCCpET14b-λCE6-HMS174−1-aExpression resulted in the formation of insoluble inclusion bodies.F355WC-terminalTTC to TGGpET14b-BL21SI+1-a Expression resulted in the formation of insoluble inclusion bodies. Open table in a new tab Expression from the pBAD33 vector under the arabinose promoter was generally under tighter control (21Guzman L.-M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3876) Google Scholar). TheXbaI-HindIII fragment containing the entirerho gene from the pET14b was placed in pBAD33, transformed into host MG1655, and expression was induced by adding 0.2% arabinose. Expression of K352A, K298A, and R299A were obtained using the pBAD33 vector. The remaining mutants either did not grow when pBAD33 was transformed into MG1655 or did not express protein. Transforming vectors into the λ host strain HSM174, which lacked T7 RNA polymerase, and inducing expression with λ phage CE6 accomplished expression of the remaining mutant proteins from pET14b vectors R160A, R238A, R269A, R272A, K283E, R296A, R353A, and R384A. Individual colonies were grown in LB media containing 0.2% maltose and ampicillin to 0.6–1.0 A600. Expression was induced by the addition of 2–4 × 109 plaque-forming units ml−1 λ phage CE6 containing the gene for the T7 RNA polymerase. Three h after infection, cells were harvested, lysed, and Rho-purified using the HiTrap metal chelating column bound with Ni2+. These mutants overexpressed protein; however, R160A, R269A, and K283E exhibited no poly(C)-dependent ATPase activity and R272A and R384A produced inclusion bodies that could not be solubilized. The poly(C)-dependent ATPase activity for the Rho mutants was measured as a function of ATP concentrations (Table II). All of the mutant proteins exhibited a decrease in kcat for ATP hydrolysis when compared with wild-type. Wild-type kcat was 2380 min−1, and only R296A approached this value at 2000 min−1. The other mutants showedkcat values one-half or less of wild-type. Three mutants (R160A, R269A, and K283E) were partially soluble but had no ATP hydrolysis activity. The Km for His-tagged wild-type was ∼5-fold higher than we reported for wild-type Rho (20Park H. Zhang X. Moon H. Zwiefka A. Cox K. Gaskell S.J. Widger W.R. Kohn H. Arch. Biochem. Biophys. 1995; 323: 447-454Crossref PubMed Scopus (39) Google Scholar). This value was consistently observed throughout these experiments. Wild-type Rho exhibited a Km(ATP) of 57 μm, whereas the remaining mutants had either similar (R296A, R299A, K352A, and R353A) or lower (R212G, R347G, and K348A)Km values ranging from 14 to 36 μm, except for K336A, which gave a Km(ATP)of 125 μm. With the exception of R296A and K336A, amino acid changes at these positions did not adversely affect ATP binding but substantially affected ATP hydrolysis.Table IIKinetic paramaters of mutant Rho proteinsRhoPoly(C)-dependent ATPaseRibo(C10)-dependent ATPaseTermination efficienciesKm(ATP)kcatKm(Ribo(C)10)kcatμmmin−1μmmin−1%Wild-type5723809850100R212G1526633343514R238A2516741037R296A542000500111053K298A364445410032R299A5783333315423K336A1251330R347G142085.616141K348A1422228680030K352A5410001000100050R353A425881231297 Open table in a new tab The mutations R347G, K348A, K352A, R353A, and F355W were clustered on or near the H-helix close to the adenosine-binding site for ATP. Mutations R347G, K348A, K352A, and R353A showed a 50–90% loss inkcat for poly(C)-dependent ATP hydrolysis, whereas the Km for ATP was similar to or lower than wild-type. ATP hydrolysis rates are induced by binding RNA at the secondary binding/tracking site (3Richardson J.P. J. Biol. Chem. 1982; 257: 5760-5766Abstract Full Text PDF PubMed Google Scholar, 19Magyar A. Zhang X. Abdi F. Kohn H. Widger W.R. J. Biol. Chem. 1999; 274: 7316-7324Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 30Pereira S. Platt T. J. Mol. Biol. 1995; 251: 30-40Crossref PubMed Scopus (21) Google Scholar). Amino acid substitutions that alter RNA binding at the secondary/tracking site were expected to alter the kinetics of ATP hydrolysis. RNA activation of ATP hydrolysis activity was determined by varying the ribo(C)10 concentrations in the presence of saturating concentrations of poly(dC) and ATP. Most mutant proteins exhibited decreased kcat values compared with the wild-type value of 850 min−1 (Table II), and the decrease in the kcat for ribo(C)10 activation generally mirrored thekcat values determined in the poly(C)-dependent ATPase assay. Of significance, R296A, K348A, and K352A showed kcat values similar with wild-type. The Km(ribo(C)10) values were sensitive to alanine screening. TheKm(ribo(C)10) for wild-type Rho was 9.0 μm. The K336A mutation showed no detectable poly(dC)-ribo(C)10-stimulated ATP hydrolysis activity, and the poly(C)-dependent ATPase activity was diminished nearly 18-fold. This was the only mutation with a large increase in theKm(ATP), suggesting that Lys336 has a direct role in ATP binding. This finding supports the notion that residues from neighboring subunits can influence ATP binding across the subunit interface (31Vincent F. Widger W.R. Openshaw M. Gaskell S.J. Kohn H. Biochemistry. 2000; 39: 9067-9076Crossref PubMed Scopus (11) Google Scholar). By comparison, most of the active mutants exhibited Km(ribo(C)10) values 5–100-fold greater than wild-type Rho, with the only exception being R347G, which had a Km(ribo(C)10) value of 5.6 μm. R296A and K352A stand out as unusual, thekcat for poly(dC)-ribo(C)10-dependent ATP hydrolysis exceeded that of wild-type yet the Km for ribo(C)10 was 500 and 1000 μm, respectively, which was 50–100-fold greater than wild-type Rho. A large increase in the Km(ribo(C)10) coupled with a slight increase in kcat for ATP hydrolysis suggest that the on-rate for ribo(C)10 was lower. To support this notion transcription termination efficiencies should be considerably less for these mutants than for wild-type. In vitro transcription termination reactions were measured using the modified trpoperon fragment (22Wu A.M. Christie G.E. Platt T. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2913-2917Crossref PubMed Scopus (81) Google Scholar), as reported (6Magyar A. Zhang X. Kohn H. Widger W.R. J. Biol. Chem. 1996; 271: 25369-25374Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Fig.1A shows the transcription termination reactions of wild-type Rho, R212G, R296A, K298A, and R299A, which are located at the putative N-terminal locus of the RNA tracking site. Lane 12 shows the run-off transcript (labeledA in Fig. 1A) in the absence of Rho; lane 1 shows the transcripts in the absence of Rho but with the inclusion of 28 μm rifampicin, lane 2 shows transcript produced in the presence of wild-type Rho, and lane 3 shows the effect of 50 μm bicyclomycin on wild-type Rho. Transcription termination efficiencies as a percentage of wild-type Rho for the mutant proteins as depicted in Fig. 1, A and B, are summarized in Table II. Mutant R212G generated only 14% of the terminated transcripts compared with wild-type Rho, whereas mutants R296A, K298A, and R299A generated 53, 32, and 23% terminated transcripts, respectively. The terminated transcripts for the mutants were longer than those seen for wild-type Rho. The terminated transcripts are labeled 1, 2, and 3 in Fig. 1A. Wild-type Rho produced terminated transcripts dominated by 2 and 3 with a small amount of 1. The mutant Rho proteins predominantly produced the longer terminated transcripts 1 and 2 but little, if any 3, the shortest terminated transcript. The production of longer terminated transcripts was reminiscent of the intermediate-size transcripts seen in the titration of Rho with the antibiotic bicyclomycin (6Magyar A. Zhang X. Kohn H. Widger W.R. J. Biol. Chem. 1996; 271: 25369-25374Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and the transcripts generated from bicyclomycin-resistant Rho mutants (6Magyar A. Zhang X. Kohn H. Widger W.R. J. Biol. Chem. 1996; 271:" @default.
• W2109503950 created "2016-06-24" @default.
• W2109503950 creator A5035547619 @default.
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• W2109503950 title "Mutations in the Rho Transcription Termination Factor That Affect RNA Tracking" @default.
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