In VitroCharacterization of Transcription Termination Factor Rho fromEscherichia coli rho(nusD)Mutants

Washburn, Robert S.; Stitt, Barbara L.

doi:10.1006/jmbi.1996.0404

Cited by 17 publications

(27 citation statements)

References 45 publications

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“…A wild-type copy of rho is carried on the plasmid pPMrho, which has a temperature-sensitive replicon. This strain fails to grow at 42°C due to the loss of rho at this temperature (22). pAU2442 was introduced into BLS107(pPMrho) by electroporation and was plated at 30, 37, and 42°C.…”

Section: Methodsmentioning

confidence: 99%

rho Is Not Essential for Viability or Virulence in Staphylococcus aureus

Washburn

Marra

Bryant

et al. 2001

Antimicrob Agents Chemother

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We have identified the gene for transcription termination factor Rho in Staphylococcus aureus. Deletion of rho in S. aureus reveals that it is not essential for viability or virulence. We also searched the available bacterial genomic sequences for homologs of Rho and found that it is broadly distributed and highly conserved. Exceptions include Streptococcus pneumoniae, Streptococcus pyogenes, Mycoplasma genitalium, Mycoplasma pneumoniae, Ureaplasma urealyticum, and Synechocystis sp. strain PCC6803, all of which appear not to possess a Rho homolog. Complementation studies indicate that S. aureus Rho possesses the same activity as Escherichia coli Rho and that the Rho inhibitor bicyclomycin is active against S. aureus Rho. Our results explain the lack of activity of bicyclomycin against many gram-positive bacteria and raise the possibility that the essentiality of rho may be the exception rather than the rule.The rho gene codes for transcription termination factor Rho (19) and is essential for the viability of Escherichia coli (5). The function of Rho is to catalyze the release of RNA from a transcription complex after a Rho-dependent terminator sequence has been transcribed. Rho has an RNA-dependent NTPase activity, which is required for transcript release (10, 12), and an RNA-DNA helicase activity (1). Several Rhodependent transcription terminators in Escherichia coli and coliphage are known (4,20).Rho is a hexamer of identical 47-kDa monomers (2, 9, 11, 17). Its ability to bind RNA is thought to be conferred by residues 22 to 116 of the 419-amino-acid protein (14). The ATP binding domain is located between residues 167 and 319 (7). No activity has been assigned to the essential C terminus of Rho, although it has been speculated as being involved in subunit interactions (1,6,8).The antibiotic bicyclomycin inhibits Rho (23). With the exception of Micrococcus luteus (15, 16), gram-positive bacteria are resistant to bicyclomycin, and rho is not essential in the gram-positive bacterium Bacillus subtilis (13,21). This study reports the sequence and characterization of the rho gene in the clinically important gram-positive pathogen Staphylococcus aureus. MATERIALS AND METHODSBacteria and growth conditions. Staphylococcus aureus was propagated in either tryptic soy broth (DIFCO) or Luria broth (LB). E. coli was grown in LB. For auxotrophy measurements with S. aureus, M9 minimal medium was supplemented with arginine, cysteine, glutamate, glycine, isoleucine, leucine, methionine, proline, and valine, all at 20 g/ml. Nicotinate and thiamine were added at 0.2 g/ml. All cultures were routinely incubated at 37°C. All strains used in this study are described in Table 1.Identification of the S. aureus rho gene. The rho gene from Staphylococcus aureus was identified from an S. aureus genomic sequence database by homology with E. coli rho. The plasmid pAU2442 used in this study is from a genomic library used in the sequencing project.Deletion mutagenesis. A rho deletion was introduced into S. aureus RN4220. This was achieved by ...

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Section: Methodsmentioning

confidence: 99%

rho Is Not Essential for Viability or Virulence in Staphylococcus aureus

Washburn

Marra

Bryant

et al. 2001

Antimicrob Agents Chemother

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“…Ligand-binding Sites on Rho-Although both molecular genetics and electrospray mass spectrometry measurements indicate that Rho has six identical subunits (57), only three ATP-binding sites are detected. As suggested by Stitt (15), these results may indicate that ATP binding to some subunits of Rho prevents ATP binding to other subunits at the same time or may indicate pre-existing asymmetry of the enzyme such that only 3 ATP molecules can bind simultaneously.…”

Section: Catalytic Cooperativity Is Consistent With Inactivationmentioning

confidence: 99%

Sequential Hydrolysis of ATP Molecules Bound in Interacting Catalytic Sites of Escherichia coli Transcription Termination Protein Rho

Stitt

1998

Journal of Biological Chemistry

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Escherichia coli transcription termination protein Rho aids in the release of newly synthesized RNA from paused transcription complexes (reviewed in Ref. 1). The homohexameric protein binds nascent RNA and, with the RNA-dependent hydrolysis of ATP, disrupts the ternary transcription complex, releasing product RNA and allowing RNA polymerase to recycle. The discovery of a 5Ј 3 3Ј RNA-DNA helicase activity of Rho (2) suggested that Rho might disrupt the RNA-DNA duplex of the transcription bubble. Recent studies of ternary transcription complexes (Refs. 3-5 and reviewed in Ref. 6) suggest that such disruption could be important in transcription termination, as could be the release of the nascent RNA just 5Ј of the RNA-DNA duplex from its interactions with RNA polymerase. As described by Nudler et al. (7), the interaction of RNA with RNA polymerase immediately 5Ј from the RNA-DNA hybrid may control the opening and closing of an RNA polymerase clamp around the DNA template near the leading edge of the enzyme, and contribute to the stability of the ternary transcription complex. An appealing model for Rho is one in which the enzyme binds to exposed mRNA behind RNA polymerase and travels 5Ј 3 3Ј along the RNA as it hydrolyzes ATP, binding and releasing RNA from different parts of the hexamer to accomplish movement (8). Such activity could release nascent RNA from RNA polymerase-binding sites and could constitute the basis for its RNA-DNA helicase activity, both of which might be involved in transcript release from paused ternary transcription complexes. The finding that the same number of ATP molecules per RNA length is hydrolyzed by Rho traveling along RNA and Rho unwinding RNA-DNA hybrids (8) supports this hypothesis.Rho binds single-stranded RNA, showing preferred entry regions on RNA upstream of eventual transcription termination sites. However, the characteristics of these regions, beyond low secondary structure and some preference for a C-rich, G-poor base composition (9), are too poorly understood to permit their identification by sequence inspection. When bound to RNA, Rho protects 80 bases from ribonuclease degradation (10, 11). The binding of Rho to 10-base RNA oligomers was reported as best fit by three tight and three weaker sites per hexamer (12, 13).The RNA-dependent hydrolysis of ATP is essential for the transcription termination function of Rho. Two components of ternary transcription complexes, the DNA template and RNA polymerase, are not required to elicit this ATPase activity, thus considerably simplifying study of the reaction (14). The reaction is particularly well stimulated by the RNA homopolymer poly(C), and Rho is frequently assayed in vitro by measuring its poly(C)-dependent ATPase activity. Previous work has shown that the Rho hexamer binds three molecules of MgATP in a single class of catalytically competent sites (15,16). An additional class of three ATP-binding sites of lower affinity has also been suggested (16), although the catalytic activity of these sites was not assessed. The stoic...

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“…We also constructed R221A to see the effect of the amino acid change. Previously, some Rho mutants were reported that displayed enhanced as well as early termination activities (14,15). From that group, we chose the mutants P103L and P235H as their altered properties were most prominent.…”

Section: Resultsmentioning

confidence: 99%

Molecular Basis of NusG-mediated Regulation of Rho-dependent Transcription Termination in Bacteria

Valabhoju

Agrawal

Sen

2016

Journal of Biological Chemistry

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The bacterial transcription elongation factor NusG stimulates the Rho-dependent transcription termination through a direct interaction with Rho. The mechanistic basis of NusG dependence of the Rho function is not known. Here, we describe Rho* mutants I168V, R221C/A, and P235H that do not require NusG for their termination function. These Rho* mutants have acquired new properties, which otherwise would have been imparted by NusG. A detailed analyses revealed that they have more stable interactions at the secondary RNA binding sites of Rho, which reduced the lag in initiating its ATPase as well as the translocase activities. These more stable interactions arose from the significant spatial re-orientations of the P, Q, and R structural loops of the Rho central channel. We propose that NusG imparts similar conformational changes in the central channel of Rho, yielding faster isomerization of the open to the closed hexameric states of the latter during its RNA-loading step. This acceleration stabilizes the Rho-RNA interactions at many terminators having suboptimal rut sites, thus making Rho-NusG interactions so essential in vivo. Finally, identification of the NusG binding sites on the Rho hexamer led us to conclude that the former exerts its effect allosterically.At the end of a significant number of operons in Escherichia coli, DNA sequences do not code for intrinsic termination signals, characterized by a hairpin and a U-stretch (1). Transcription termination factor Rho, a homo-hexameric RNA-dependent ATPase, induces termination in these operons. Rho loads onto unstructured C-rich RNA sequences (rut sites, the Rhoutilization sites) and is capable of translocating along the RNA using its RNA-dependent ATPase activity, and eventually dislodges the elongating RNA polymerase (RNAP) 3 (1, 2). By virtue of these properties of Rho, long stretches of untranslated RNA sequences are its ideal targets. Transcription on these stretches can face premature termination by Rho, which usually happens in the cases of transcription-translation uncoupling. Recent genomics study has revealed that Rho-dependent termination is widespread in vivo, which could be the main reason for its essentiality (3-6).NusG, a transcription elongation factor, being bound to the elongation complex (EC) through its N-terminal domain (NTD) helps the latter to overcome the pauses by increasing the elongation rate (5, 7, 8). Its C-terminal domain (CTD) is capable of interacting with the Rho (9). This interaction stimulates the Rho-dependent termination process in vitro, which is manifested as early termination (10, 11). More recent studies indicated that NusG is essential for recruiting Rho on a subset of terminators in vivo (4, 6). Mechanism of stimulation of Rho-dependent termination by NusG in vitro as well as the molecular basis of NusG dependence of Rho to utilize a subset of terminators in vivo is not known, knowledge of which is essential to understand the intricacies of this transcription termination process.In this study, by screening a library of ...

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In VitroCharacterization of Transcription Termination Factor Rho fromEscherichia coli rho(nusD)Mutants

Cited by 17 publications

References 45 publications

rho Is Not Essential for Viability or Virulence in Staphylococcus aureus

rho Is Not Essential for Viability or Virulence in Staphylococcus aureus

Sequential Hydrolysis of ATP Molecules Bound in Interacting Catalytic Sites of Escherichia coli Transcription Termination Protein Rho

Molecular Basis of NusG-mediated Regulation of Rho-dependent Transcription Termination in Bacteria

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