Nucleotide Binding Induces Conformational Changes in Escherichia coli Transcription Termination Factor Rho

Abstract

The Escherichia coli Rho protein uses the energy of ATP binding and hydrolysis to translocate along RNA and cause transcription termination. Using fluorescence stopped-flow kinetic studies, we have discerned the conformational changes in the Rho protein that occur upon nucleotide and nucleic acid binding. We show that the 2,(3)-O-[N-methylanthraniloyl] derivative of ATP (mant-ATP) is a good fluorescent substrate of Rho and is hydrolyzed with a K m comparable with that for ATP but a k cat five to six times slower than that for ATP. The kinetics of ATP and mant-ATP binding indicates that, in the absence of RNA, the Rho protein is structurally distinct from the Rho hexamer found when bound to RNA or DNA. In the absence of RNA, the nucleotidebinding rates are 50-to 70-fold slower, and the dissociation rates are 40-to 120-fold slower than the corresponding rates in the presence of RNA. We conclude that RNA or DNA binding to the primary nucleic acid binding sites causes conformational changes in the Rho hexamer that result in the opening of the subunit interfaces. Furthermore, the kinetic studies revealed a unique protein conformational change in the Rho⅐RNA complex upon ATP binding that is a result of RNA contacting the secondary nucleic acid binding sites in the central channel of the Rho ring. This conformational change seems to render the Rho ring competent in ATP hydrolysis and translocation.The Escherichia coli transcription termination factor Rho is a hexameric protein that has the ability to unwind RNA/DNA heteroduplexes (1-5). Although the exact mechanism by which Rho causes transcription termination is not known, Rho protein binds nascent mRNA at specific loading sites, and it is believed that Rho translocates along RNA until it reaches the transcription complex, where it disrupts the transcription ternary complex (3, 6). Translocation along nucleic acid is, therefore, a basic activity of Rho similar to helicases (7, 8), and understanding it requires the knowledge of how nucleotide binding and hydrolysis events at the multiple nucleotide-binding sites on the Rho hexamer are coordinated to the mechanics of protein translocation. In this study, we measured the kinetics of nucleotide binding to Rho complexed to RNA or DNA that reveal conformational changes in the Rho protein that we believe are important for RNA binding and ATP hydrolysis.E. coli Rho protein contains two types of nucleic acid binding sites, referred to as the primary and the secondary sites (9). The primary nucleic acid binding sites reside in the N-terminal domains that form a crown over the Rho hexamer (10), and these sites bind pyrimidine-rich DNA or RNA with a high affinity (11). In addition to wrapping around the primary sites, the RNA but not the DNA interacts with the secondary sites in the central channel of the hexamer (12). The kinetics of RNA binding to the Rho hexamer showed that the initial binding of RNA occurs at a diffusion-limited rate constant to the primary sites (13). The primary sites act as loading sites that facilitate th...

ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses

“…To show that the noncanonical G4 motif of OsYchF1 ( 230 NMSE 233 ) is essential for ATP binding, site-directed mutagenesis was performed to convert this motif to the canonical G4 motif of other P-loop GTPases (i.e., NKSD), and the mutant was designated as OsYchF1-G4′. We performed titration experiments with 2′,(3′)-O-[N-methylanthraniloyl] derivatives of ATP (Mant-ATP) and GTP (Mant-GTP), fluorescent analogs of ATP and GTP, respectively, which are commonly used in nucleotide-binding measurements (13). In this assay, OsYchF1 can bind to both GTP and ATP with similar affinities (Fig.…”

Cooperative Mechanism of RNA Packaging Motor

“…the MANT group linked to the 2Ј-hydroxyl versus the 3Ј-hydroxyl group). Such an effect has been observed for MANT-ATP binding to the transcription termination factor Rho (8). Therefore, we investigated the interactions of P4 with two additional MANT-nucleotide derivatives, where the fluorophore was attached specifically to the 3Ј-hydroxyl (3Ј-MANT-2Ј-dATP) or the 2Ј-hydroxyl (2Ј-MANT-3Ј-dATP) of the deoxyribose ring.…”

“…This process requires a portal complex that operates as the molecular motor and converts chemical energy into mechanical work. Doublestranded RNA bacteriophages from the Cystoviridae family (6)(7)(8)(9)(10)(11)(12)(13)(14) package their ssRNA 1 genomic precursors using a hexameric portal complex, the packaging NTPase P4 (1). P4 proteins share sequence and structural similarities with hexameric helicases and some of them possess helicase activity (2,3).…”

Ligand-induced and small-molecule control of substrate loading in a hexameric helicase

“…Many hexameric motors undergo substrate-dependent conformational changes that couple activity to the productive binding of client substrates (1)(2)(3)(4). Internal regulatory domains and exogenous proteins or small molecules frequently impact client substrate recruitment and engagement by these enzymes (5)(6)(7)(8); however, it is generally unclear how such factors control helicase or translocase dynamics.…”

“…It is well established that the sequence of the RNA itself has a pronounced impact on whether a transcript will be acted upon by Rho (12,(27)(28)(29). Binding of pyrimidine-rich sequences to the N-terminal RNA-binding domains of Rho is a particularly well-known accelerant of Rho's ATPase activity (30), with presteady-state ATPase assays showing that the formation of a catalytically competent Rho ring is governed by a rate-limiting RNA-and ATP-dependent conformational change (8). The liganddependence of this isomerization event correlates with the requirements for ring closure identified in an accompanying study (16).…”