exist within the central cavity of the closed ring
termination factor has remained elusive, it has beenwell established that Rho binds preferentially to
transcript is annealed to a DNA handle attached to a
ultra-stable dual optical trap system capable ofapplying a wide range of stretching forces and
oltp :unstructured C-rich RNA at the primary bindingsites.9 The pyrimidine-rich sites of Rho interactionwithin natural RNA transcripts, termed Rho utili-zation (rut) sites, are typically ~80 nucleotides inlength.1012
detecting sub-nanometer structural transitions.14 Bymoving the two traps with respect to each other, theauthors apply a precisely calibrated stretching forceto the system and visualize the extension as afunction of the applied force in real time in so-calledconformation of the Rho hexamer.5,8 Although aclear consensus sequence for the loading of Rho
separate bead, creating a molecular dumbbell.This dumbbell is then held on each end using anCOMM
Getting a Grip o
In bacteria, regulation of transcriptional termina-tion is carried out by two general mechanisms:intrinsic and factor-dependent termination.1 Infactordependent termination, a molecular machinecalled Rho loads onto the nascent RNA andmechanically forces the RNA polymerase (RNAP)elongation complex (EC) to dissociate. Rho termi-nation factor is a homohexameric toroidal proteincomplex that couples the energy of ATP bindingand hydrolysis to translocation in the 53direction along nascent RNA transcripts. Since thediscovery of Rho-dependent termination in the late1960s,2 a combination of biochemical and structuralanalyses has provided a detailed, albeit incomplete,view of the molecular mechanism of the Rhotermination factor. Structural analysis of Rho byelectron microscopy and X-ray crystallography hasprovided strong evidence that the Rho hexamer canexist in two general conformational states: an openoligomeric form resembling a lock washer and atopologically closed ring structure.35 There aretwo distinct types of RNA binding sites within thecomplex. Each of the Rho monomer units withinthe hexamer may interact with RNA at what hasbeen termed the primary binding site,6,7 while asecondary RNA binding site has been shown to
Contents lists available
Journal of Mj ourna l homepage: htRho termination factor is among the most power-ful molecular machines to be characterized, capableof generating forces in excess of 200pN.13 Thus, themolecular details of Rho action has garnered interestnot only from researchers focused on mechanisms oftranscriptional regulation but also from a largecommunity of biophysicists interested in the mech-anochemistry (the use of chemical energy to perform
0022-2836/$ - see front matter 2012 Elsevier Ltd. All rights reserveTARY
mechanical work) of biological motor proteins. Ofparticular interest is understanding how the Rhohexamer binds to rut and subsequently translocatestoward the active RNAP molecule. Two classes ofmodels for Rho translocation have been proposed:tethered tracking and rut-free tracking (or simpletranslocation).1 In the tethered tracking model, Rhomaintains contacts with the primary sites of RNAinteraction established at the rut site during initialbinding, and movement of the nascent RNAtranscript at the secondary RNA interaction sitewithin the central cavity of the Rho hexamerduring processive translocation generates a loopof RNA. In contrast, a rut-free tracking modelposits release of the RNA from the primary bindingsites and a simple translocation of the hexamer atthe secondary site within the central pore of thecomplex.In this issue of the Journal of Molecular Biology,
Koslover et al. employ an elegant singlemoleculeassay to directly probe the interaction of Rho withRNA transcripts and provide strong support for atethered tracking model for Rho translocation. Withthe use of an optical trapping system, a biotinylatedstalled RNAP EC is immobilized on a streptavidincoated bead and the 5 end of the nascent RNA
J. Mol. Biol. (2012) 423, 661663
ecular Biology/ /ees .e lsev ie r.com. jmbforceextension curves (FECs). In this type ofexperiment, structural changes within a system areobserved as abrupt extension changes (rips) in theFEC, providing information about the structure andenergetics underlying the observed mechanicaltransition.To analyze the nature of Rho factorRNA in-
teractions, the authors utilized three different RNA
(1988). Interactions of Escherichia coli transcriptionsubstrates, each possessing the rut site from the tR1terminator followed by a segment of RNA thatvaried in length (30, 75, or 150 nucleotides). Initialexperiments characterizing the binding of Rho factorwith rut used the non-hydrolyzable ATP analogAMP-PNP to promote Rho binding. In the presenceof Rho factor, two distinct populations of transitions(29nm and 46nm) were observed. Based upon aseries of experiments, these two transitions weredesignated as primary rips and secondary rips,corresponding to the release of RNA from all sixprimary Rho binding sites or from the six primarysites plus the secondary binding site, respectively.Using these data, the authors estimate the bindingfootprints for the complexes underlying each of theobserved rip transitions, yielding values of 57nucleotides for binding to the six primary sites and85 nucleotides for binding to the six primary sitesplus the secondary sites. Notably, singlemoleculeanalysis of binding footprints does not suffer fromheterogeneity between individual RhoRNA com-plexes that may obscure results in ensemble foot-printing analyses.Next, the authors exploited the power of their
singlemolecule assay to directly discriminate be-tween the tethered and rut-free tracking models forRho factor translocation. In these experiments,FECs were analyzed for Rho complexes bound toeach of the three different length RNA substrates inthe presence of ATP, which allows the Rho complexto actively translocate to the stalled RNAP EC. IfRho translocation proceeds via rut-free tracking,then the expected size of the rips observed in theFECs would be on the order of a few nanometersand independent of the size of the RNA substrate.In contrast, if Rho translocates using a tetheredtracking mechanism, then the expected rip sizeswould be larger as translocation progresses furtherand should be proportional to the size of the RNAsubstrate utilized in the experiment. Strikingly, asthe length of the RNA substrate was increased, anew population of larger rips was observed,providing unambiguous support for the tetheredtracking model of Rho translocation. The observa-tion that Rho remains bound to the rut site duringtranslocation has important implications for theinterplay between the Rho termination factor andother molecular machines acting on nascent RNAtranscripts.The study reported by Koslover et al. demon-
strates the use of singlemolecule force spectroscopyin dissecting the interactions between proteinenzymes and their nucleic acid substrates. Thissingle molecule approach is ideally suited to addressmany remaining questions about Rho-dependenttermination. For example, since Rho-dependent
662termination requires the Rho complex to catchup to an actively transcribing RNAP EC, it will beinteresting to study the competing rates of thetermination factor rho with RNA. I. Bindingstoichiometries and free energies. J. Mol. Biol. 199,609622.
12. Zalatan, F. & Platt, T. (1992). Effects of decreasedcytosine content on rho interaction with the rho-dependent terminator trp t in Escherichia coli. J. Biol.Chem. 267, 1908219088.
13. Schwartz, A., Margeat, E., Rahmouni, A. R. &Boudvillain, M. (2007). Transcription terminationfactor rho can displace streptavidin from biotinylatedRNAP EC and Rho terminator translocation, aphenomenon referred to as kinetic coupling.15 Inaddition, the potential to directly analyze the impactof actively translating ribosomes and other trans-acting factors such as the NusG protein on theefciency of Rho-dependent termination promisesto yield additional insights into the molecularmechanism of transcriptional regulation.
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Landick, R. & Block, S. M. (2005). Direct observation