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

Abstract

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...

The Mechanism of ATP Hydrolysis at the Noncatalytic Sites of the Transcription Termination Factor Rho

“…Only one ATP is hydrolyzed at a time at the catalytic site. This rate is estimated at Ͼ240 s Ϫ1 from this study as well as a published study on the ATPase kinetics (12). In that report (12), a pre-steady-state burst of ATP hydrolysis was observed, implying that a step following ATP hydrolysis is a rate-limiting step.…”

“…This rate is estimated at Ͼ240 s Ϫ1 from this study as well as a published study on the ATPase kinetics (12). In that report (12), a pre-steady-state burst of ATP hydrolysis was observed, implying that a step following ATP hydrolysis is a rate-limiting step. After the rate-limiting step, the next catalytic subunit presumably goes through the same cycle of ATP binding and hydrolysis.…”

“…The amino acid sequence homology between rho and the ␤ subunit is more striking than the amino acid sequence homology between the ␣ and ␤ subunits of the F 1 -ATPase protein (11). In addition to the structural similarity, the ATPase mechanisms of both rho and the T7 gene 4 helicase show striking similarity to the binding change mechanism of the F 1 -ATPase protein (9,12).…”

“…Combined with the recently published model of ATPase reaction at the catalytic site (12), it allows us to make a comparison between the ATPase reaction pathway at the catalytic versus the noncatalytic sites (Fig. 4).…”

Kinetic Pathway of dTTP Hydrolysis by Hexameric T7 Helicase-Primase in the Absence of DNA

“…11), E. coli DnaB helicase hydrolyzes three-four ATPs simultaneously (12), and RuvB helicase hydrolyzes two ATPs (25). In the presence of RNA, Rho protein has been shown to hydrolyze one to three ATPs per hexamer (26,27). Similarly, sequential hydrolysis of NTP has been proposed for T7 helicase in the absence of DNA (11) and for Rho protein in the presence of RNA (27).…”