A physical model for the translocation and helicase activities of Escherichia coli transcription termination protein Rho.

Geiselmann, Johannes; Wang, Yan; Seifried, Steven E.; Hippel, Peter H. von

doi:10.1073/pnas.90.16.7754

Cited by 97 publications

(81 citation statements)

References 44 publications

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“…This model differs from two others that have been proposed recently (47,48) in ascribing a major role to a part of the protein that has not previously been implicated. Although the parts of Rho that comprise loop R, helix G, and ␤-strand 8 have not been shown yet to interact directly with RNA, the corresponding segments of RecA, another protein with a core ATP-binding domain that is topologically identical to that of F 1 -ATPase (23,49), make direct contact with DNA.…”

Section: Functional Changes Involving Non-conserved Residuescontrasting

confidence: 49%

Structural Organization of Transcription Termination Factor Rho

Richardson

1996

Journal of Biological Chemistry

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Escherichia coli has two known modes for termination of RNA transcription (1-4). One is intrinsic to the function of RNA polymerase, which can spontaneously terminate transcription in response to certain, limited sequences. The other mode is dependent upon the action of an essential protein factor called Rho and occurs at sequences that are specific for its function but that are less constrained than the sequences for intrinsic termination.Rho protein functions as a hexamer of a single polypeptide chain with 419 residues, which is the product of the rho gene (5). It is an RNA-binding protein with the capacity to hydrolyze ATP and other nucleoside triphosphates. Rho acts to cause termination by first binding to a site on the nascent transcript and by subsequently using its ATP hydrolysis activity as a source of energy to mediate dissociation of the transcript from RNA polymerase and the DNA template (6). In the cell, the ability of Rho to act at several terminators is dependent upon the presence of an essential 21-kDa protein called NusG (7) that binds both RNA polymerase and Rho itself (8). In vitro the dependence on NusG became apparent only at proximal terminators (at sites Ͻ300 base pairs from the promoter) and under conditions when the RNA molecules are being elongated at the in vivo rate of ϳ40 -50 nucleotides/s (9). The requirement for NusG when RNA chain growth is fast suggests that the NusG is acting to overcome a kinetic limitation of Rho to act alone, perhaps through mediating earlier access to the nascent RNA by the formation of a complex of Rho with RNA polymerase (10).The mechanism of how Rho acts to dissociate the transcription complex is unknown. One important approach to elucidating how interactions with RNA mediate termination of transcription is to determine the structure of the protein. Until a good crystal structure becomes available the properties of its structure will have to be inferred from other, less direct methods, such as biochemical characterization of the protein, phylogenetic comparative analyses, and the functional properties of mutants with known amino acid changes. This review summarizes our current understanding of the structure and function of transcription termination factor Rho based on these indirect approaches.

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Section: Functional Changes Involving Non-conserved Residuescontrasting

confidence: 49%

Structural Organization of Transcription Termination Factor Rho

Richardson

1996

Journal of Biological Chemistry

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“…This binding activates an RNAdependent ATPase of rho that fuels the directional translocation (5' -+ 3') of the rho hexamer along the nascent RNA ( [47,48]; Katherine Walstrom, unpublished data). Termination is thought to be triggered by the intrinsic ATP-dependent RNA-DNA helicase activity of rho [49] when it reaches the transcription complex paused at putative rho-dependent terminators along the template [50-521. Thus the specific positions of rho-dependent terminators along the template (as well as the efficiency of the rho-dependent termination process) are controlled by the kinetic coupling of the relative rates of the translocation of rho along the nascent RNA and of polymerase along DNA template ([53]; see also [37] and Fig.…”

Section: Cis Effects As Regulatory Elements In Transcript Elongation mentioning

confidence: 99%

Specificity mechanisms in the control of transcription

Hippel

Rees

Rippe

et al. 1996

Biophysical Chemistry

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In this overview we analyze and illustrate the principles underlying some of the specificity mechanisms that control the initiation, elongation, and termination phases of transcription. Thermodynamic mechanisms dominate in the first steps of initiation, where promoters at various levels of activation can be considered to be in competition for a limiting supply of core RNA polymerase. In the later stages of initiation, as well as in elongation and termination, the regulatory mechanisms that control specificity are largely kinetic, involving rate competition between branching reaction pathways where the outcome depends on the rates (and equilibria) of reaction and interconversion of different forms of the transcription complex. Elongation complexes are very stable at most positions along the DNA template, meaning that only RNA chain elongation (and editing) can occur at these positions. However, the stability of transcription complexes decreases abruptly when termination sequences are encountered, and here the outcome can be easily switched between elongation and termination (RNA release) by minor changes in the relative rates of these competing processes. Cis effecters, defined as sites at which regulatory proteins bind to upstream activation loci on either the DNA or the nascent RNA, play important roles in the control of both initiation and of the elongation-termination decision. Examples, drawn from studies of phage A N-dependent antitermination and E. coli rho-dependent termination processes, illustrate the flexibility and additivity of regulatory components within control mechanisms in transcription that involve multiple determinants. The generality of such regulatory principles are stressed.

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“…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.…”

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confidence: 99%

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

Jeong

Kim

Patel

2004

Journal of Biological Chemistry

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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...

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A physical model for the translocation and helicase activities of Escherichia coli transcription termination protein Rho.

Cited by 97 publications

References 44 publications

Structural Organization of Transcription Termination Factor Rho

Structural Organization of Transcription Termination Factor Rho

Specificity mechanisms in the control of transcription

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

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