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Rho Termination Factor

LaDean Cooley '09 and Piero Sanfilippo '09


I. Introduction

 Escherichia coli has two known modes for termination of RNA transcription. 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 [3].

Rho protein functions as a hexamer of a single polypeptide chain with 419 residues, which is the product of the rho gene. 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 from the transcription holoenzyme 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

 Its six identical subunits are arranged in a torus with six canonical ATP binding sites located at the interfaces of the C-terminal domains of adjacent subunits. Each ATP binding site is composed of adjacent Walker-A/Walker-B motifs typical of RecA-like proteins that utilize NTP to perform mechanical work in the cell  After binding to a transcript at a cytosine-rich rut (Rho utilization) site, Rho loads the polynucleotide into its central channel before adopting a conformation competent to processively translocate along the strand [1]. 

II. General Structure

The Rho protein is made up of six subunits that pack laterally into a hexamer . The subunits are peanut shaped and are almost identical in structure, each composed of an N-terminal domain and a C-terminal domain . The N-terminal domain consists of two subdomains: a three-helix bundle followed by a five-stranded β barrel . Each of the six C-terminal domains consists of a parallel β sheet sandwiched between several α helices . There are 3 key motifs located in the C-terminal domain. The P-loop is required for ATP binding and hydrolysis. The R-loop and the Q-loop are believed to form the secondary RNA binding site  [4] .

III. Subunit-Subunit Interaction

The hexameric conformation of Rho is held together by lateral interactions between subunits. The interactions are the same for every subunit set and occur both between the N and C terminal domains. The main interaction is attained by the packing of an α11 helix on one subunit against the β7/α8 and β8/α9 junctions on a neighboring subunit .

IV. RNA Binding 

Rho has two distinct primary nucleic acid binding sites. The primary mRNA binding sites are formed by the N-terminal domains, which have the ability to bind either single-stranded DNA or RNA. Each N-terminal domain binds a dinucleotide segment in a network of contacts that explain the preference of Rho for cytosine. The first nucleotide base packs into a hydrophobic enclosure that is formed by the side chains of Tyr 80, Glue 108, Tyr 110, and is too small to comfortably hold purine bases. For the second nucleotide the cytosine base stacks on the aromatic side chain of Phe 64, while its O2, N3, and N4 groups interact with the side chains of the neighboring Arg 66 and Asp 78  [2] . No contacts are seen to the 2´ hydroxyl of the bound nucleic acid, explaining why Rho is able to bind both ssDNA and ssRNA.  

RNA translocation and unwinding catalyzed at Rho's secondary RNA binding site, located in the C-terminal domain This function depends on two sequence motifs known Q-loop and the R-loop . Both loops line the interior hole of the hexamer.  Each Q-loop lies on the upper segment of the C-terminal domain and extends into the center of the ring. The constellation of the six Q-loops in the hexamer together form the narrowest constriction of the interior hole. R-loops are implicated in both ATP and RNA binding. Each R-loop resides on a segment located at the subunit-subunit interface between the C-terminal domains, and lies both adjacent to and above the The P-loop of the ATP binding pocket. Part of each R-loop also lines the interior hole of the Rho hexamer [4]. RNA binding to the secondary state coincides with closure of the hexameric ring and stimulation of the ATPase activity, presumably by introducing conformational changes between subunits and residues around the ATP binding site [5].  

V. Rho Function

Rho binds to RNA  (See Diagram) and then uses its ATPase activity to provide the energy to translocate along the RNA until it reaches the RNA-DNA helical region, where it unwinds the hybrid duplex structure. Each ATP-binding pocket of Rho is formed at the interface between two adjacent C-terminal domains.  All six subunits are catalytically competent and hydrolyze ATP sequentially. Translocation of RNA is driven by the weak to tight binding transition of nucleotide in the catalytic site and hydrolysis is coordinated between adjacent subunits by the transmission of stress via the catalytic arginine finger [1].  

Dynamic stresses in a ring-shaped motor protein can regulate and coordinate the hydrolysis cycle of neighboring subunits. This occurs both via interfacial interactions between subunits of the protein and via the RNA substrate during translocation. Although most of the communication occurs between adjacent subunits, stress can propagate around the ring producing weaker—but still important—effects on nonnearest neighbor subunits. That is, stress mediates both local and long-range regulation of the catalytic cycle [5].  

VI. References

1. Adelman, Y.J. Jeong, J.C. Liao, G. Patel, D.E. Kim, G. Oster and S.S. Patel, Mechanochemistry of transcription termination factor Rho, Mol. Cell 22 (2006), pp. 611–621    

2. Bogden, C.E.; Fass, D.; Bergman, N.; et al. “The structural basis for terminator recognition by the Rho transcription termination factor.” Mol. Cell. v. 3 p. 487–493. 1999.

3. Richardson, J.P., and Ruteshouser, E.C. 1986. Structrual Organization of Transcription Termination Factor Rho. J. Mol. Biol. 189, 413-419..

4. Skordalakes, E., and J. M. Berger. 2003. Structure of the Rho transcription terminator: mechanism of mRNA recognition and helicase loading. Cell 114:135-146

5. Skordalakes, E., and J. M. Berger. 2006. Structural Insights into RNA-Dependant Ring Closure and ATPase Acitivation by the Rho Terminatino Factor. Cell 127:553-564

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