Thermodynamic and enzymological characterization of the interaction between transcription termination factor .rho. and .lambda. cro mRNA

Faus, Ignacio; Richardson, John P.

doi:10.1021/bi00434a054

Cited by 45 publications

(48 citation statements)

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“…Binding to this site is a prerequisite for the activation of the ATPase activity of Rho. This is required for its subsequent helicase and transcription termination functions (14,36). Our data and previous findings suggest that the RNA sequence in the vicinity of tnaC stop codon constitutes the critical entry site for Rho.…”

Section: Discussionsupporting

confidence: 71%

Analysis of Tryptophanase Operon Expression in Vitro

Gong

Yanofsky

2002

Journal of Biological Chemistry

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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-peptidyltRNA 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 -TnaCtRNA 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.Escherichia coli and many other Gram-negative bacteria use the enzyme tryptophanase to degrade L-tryptophan to indole, pyruvate, and ammonia (1), allowing these microorganisms to utilize tryptophan as a source of carbon, nitrogen, and energy (2). The tryptophanase (tna) operon of E. coli has two major structural genes, tnaA, encoding tryptophanase, and tnaB, encoding a low affinity tryptophan permease (3, 4). Initiation of transcription of the tna operon is regulated by catabolite repression (5-7). Once initiated, continuation of transcription into the tnaA-tnaB structural gene region depends on tryptophan-induced transcription antitermination. Antitermination is achieved by some feature of induction that prevents Rho factor from terminating transcription in the leader region of the operon (5-7). The tna operon contains a 319-base pair transcribed leader region upstream of the tnaA initiation codon. The transcript of this leader region bears a coding segment, tnaC, specifying a 24-residue leader peptide, TnaC, that contains a single tryptophan residue. Synthesis of TnaC is essential for induction (3,8,9). Replacing the tnaC start codon by a stop codon (9, 10) or replacing Trp codon 12 by a codon for some other amino acid (10) prevents induction.Evidence supporting the essential role of Rho factor in mediating transcription termination in the tna operon leader region was provided by analyses of Rho mutants (8), examination of Rho-inhibiting drugs (11), and deletion of a leader region sequence-rich in C residues (10). Mutations in rho that reduce Rho factor activity as well as addition of bicyclomycin, an inhibi...

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Section: Discussionsupporting

confidence: 71%

Analysis of Tryptophanase Operon Expression in Vitro

Gong

Yanofsky

2002

Journal of Biological Chemistry

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“…Rho Binds to Circular RNA-To determine whether Rho can bind the circular cro derivative RNA, we used the nitrocellulose filter-binding technique to isolate complexes of Rho with radiolabeled RNA (15,18). With saturating levels of Rho, ϳ60% of the circular cro RNA was retained on the filter.…”

Section: Resultsmentioning

confidence: 99%

“…Faus and Richardson (15) showed that blocking this portion of the rut site with an oligonucleotide inhibited Rho's ability to bind to cro RNA. Also, Chen et al (20) showed that blocking the rut site with a similar DNA oligonucleotide inhibited Rho's in vitro termination activity at the tR1 terminator of the cro gene.…”

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confidence: 99%

Transcription Factor Rho Does Not Require a Free End to Act as an RNA-DNA Helicase on an RNA

Burgess

Richardson

2001

Journal of Biological Chemistry

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Escherichia coli Rho factor is a ring-shaped, homohexameric protein that terminates synthesis of RNA through interactions with the nascent RNA transcript. Because its mechanism of action may involve translocation of the RNA transcript through the hole in its ring structure, its action could depend on the availability of a free 5 terminus. To determine whether Rho's activity is 5-end-dependent, its ability to bind to and function on a circular derivative of cro mRNA was investigated. The circular derivative was made in vitro by action of RNA ligase on a derivative of cro RNA containing an extra 10-nucleotide sequence near the 5-end that was complementary to a sequence located near the 3-end. Rho bound nearly as tightly to the circular derivative RNA as to the standard cro transcript. Rho was also able to readily dissociate a DNA oligonucleotide from its helical complex with the circular RNA in an ATP-dependent reaction. Thus, the action of Rho on a transcript does not depend on the availability of a free 5 terminus.Transcription termination factor Rho is an RNA-binding protein that couples the energy derived from ATP hydrolysis to actions that dissociate a RNA transcript from its biosynthetic complex with DNA and RNA polymerase (1, 2). Rho also can dissociate a RNA molecule bound to DNA through a short hybrid helix in a reaction that is dependent on ATP hydrolysis and the presence of an attachment site for Rho on the RNA on the 5Ј side of the hybrid helix (3-5). This RNA-DNA helicase activity may mimic the process of removal of the nascent RNA strand from the transcription elongation complex.Rho factor is believed to function in vivo as a hexamer consisting of six polypeptide subunits arranged in a ring-shaped structure (6, 7). Burgess and Richardson (8) have recently presented a model for the Rho-RNA complex in which a segment of RNA, called a rut site (Rho utilization site), binds to a cleft comprised of the N-terminal RNA-binding domains of the six individual subunits located at one end of the hexameric structure (the crown) (9 -11). RNA sequence 3Ј of the rut site then passes into the hole located at the center of the hexamer. Evidence for passage of a single-stranded nucleic acid substrate through a ring-shaped hexameric structure has been observed for other hexameric helicases, such as DnaB and the T7 gene 4 product (12, 13).The process by which Rho binds to and captures the 3Ј-end of an RNA in the center of the hexamer is not known. Gan and Richardson (14) have recently presented data indicating that Rho, in vitro, could form hexamers by partial assembly of subunits on an RNA. Another mechanism could have the RNA enter into the center of the hexameric structure through an opening or notch in the ring (7). A third mechanism may involve Rho threading onto the nascent transcript from the free 5Ј-end of the RNA. To test the model in which Rho requires a free end of RNA to thread onto in order to function as a terminator, we investigated Rho's ability to utilize its ATP-dependent helicase activity on an RNA lac...

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“…Rho carries an RNA-dependent ATPase activity that displays an RNA cofactor requirement similar to that of rho-dependent termination: i.e., a rho binding site on the RNA that is relatively devoid of stable secondary structure and relatively rich in cytosine residues. This ATPase activity and its cofactor requirements have been extensively studied (Lowery & Richardson, 1977a,b;Galluppi & Richardson, 1980;McSwiggen, 1985;von Hippel et al, 1987;Faus & Richardson, 1989). Using this RNA-dependent ATPase as an energy source, Brennan et al (1987) have shown that rho can act as a 5' 4 3' directional RNA-DNA helicase on RNA-DNA heteroduplex constructs that carry a singlestranded region of RNA at the 5' end.…”

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confidence: 99%

Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA

1992

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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], where n 2 6). Rho binds to long RNA chains as a hexamer characterized by D3 symmetry. Each hexamer binds -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), 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.

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Thermodynamic and enzymological characterization of the interaction between transcription termination factor .rho. and .lambda. cro mRNA

Cited by 45 publications

References 32 publications

Analysis of Tryptophanase Operon Expression in Vitro

Analysis of Tryptophanase Operon Expression in Vitro

Transcription Factor Rho Does Not Require a Free End to Act as an RNA-DNA Helicase on an RNA

Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA

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