3
C OMMENTARY Getting a Grip on the Terminator In bacteria, regulation of transcriptional termina- tion is carried out by two general mechanisms: intrinsic and factor-dependent termination. 1 In factordependent termination, a molecular machine called Rho loads onto the nascent RNA and mechanically forces the RNA polymerase (RNAP) elongation complex (EC) to dissociate. Rho termi- nation factor is a homohexameric toroidal protein complex that couples the energy of ATP binding and hydrolysis to translocation in the 53direction along nascent RNA transcripts. Since the discovery of Rho-dependent termination in the late 1960s, 2 a combination of biochemical and structural analyses has provided a detailed, albeit incomplete, view of the molecular mechanism of the Rho termination factor. Structural analysis of Rho by electron microscopy and X-ray crystallography has provided strong evidence that the Rho hexamer can exist in two general conformational states: an open oligomeric form resembling a lock washer and a topologically closed ring structure. 35 There are two distinct types of RNA binding sites within the complex. Each of the Rho monomer units within the hexamer may interact with RNA at what has been termed the primary binding site, 6,7 while a secondary RNA binding site has been shown to exist within the central cavity of the closed ring conformation of the Rho hexamer. 5,8 Although a clear consensus sequence for the loading of Rho termination factor has remained elusive, it has been well established that Rho binds preferentially to unstructured C-rich RNA at the primary binding sites. 9 The pyrimidine-rich sites of Rho interaction within natural RNA transcripts, termed Rho utili- zation (rut) sites, are typically ~80 nucleotides in length. 1012 Rho termination factor is among the most power- ful molecular machines to be characterized, capable of generating forces in excess of 200 pN. 13 Thus, the molecular details of Rho action has garnered interest not only from researchers focused on mechanisms of transcriptional regulation but also from a large community of biophysicists interested in the mech- anochemistry (the use of chemical energy to perform mechanical work) of biological motor proteins. Of particular interest is understanding how the Rho hexamer binds to rut and subsequently translocates toward the active RNAP molecule. Two classes of models for Rho translocation have been proposed: tethered tracking and rut-free tracking (or simple translocation). 1 In the tethered tracking model, Rho maintains contacts with the primary sites of RNA interaction established at the rut site during initial binding, and movement of the nascent RNA transcript at the secondary RNA interaction site within the central cavity of the Rho hexamer during processive translocation generates a loop of RNA. In contrast, a rut-free tracking model posits release of the RNA from the primary binding sites and a simple translocation of the hexamer at the secondary site within the central pore of the complex. In this issue of the Journal of Molecular Biology, Koslover et al. employ an elegant singlemolecule assay to directly probe the interaction of Rho with RNA transcripts and provide strong support for a tethered tracking model for Rho translocation. With the use of an optical trapping system, a biotinylated stalled RNAP EC is immobilized on a streptavidincoated bead and the 5end of the nascent RNA transcript is annealed to a DNA handle attached to a separate bead, creating a molecular dumbbell. This dumbbell is then held on each end using an ultra-stable dual optical trap system capable of applying a wide range of stretching forces and detecting sub-nanometer structural transitions. 14 By moving the two traps with respect to each other, the authors apply a precisely calibrated stretching force to the system and visualize the extension as a function of the applied force in real time in so-called forceextension curves (FECs). In this type of experiment, structural changes within a system are observed as abrupt extension changes (rips) in the FEC, providing information about the structure and energetics underlying the observed mechanical transition. To analyze the nature of Rho factorRNA in- teractions, the authors utilized three different RNA http://dx.doi.org/10.1016/j.jmb.2012.09.004 J. Mol. Biol. (2012) 423, 661663 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter © 2012 Elsevier Ltd. All rights reserved.

Getting a Grip on the Terminator

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http://dx.doi.org/10.1016/j.jmb.2012.09.004 J. Mol. Biol. (2012) 423, 661–663

Contents lists available at www.sciencedirect.com

Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

COMMENTARY

Getting a Grip on the Terminator

In bacteria, regulation of transcriptional termina-tion is carried out by two general mechanisms:intrinsic and factor-dependent termination.1 Infactor‐dependent 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 5′→3′direction 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.3–5 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 toexist within the central cavity of the closed ringconformation of the Rho hexamer.5,8 Although aclear consensus sequence for the loading of Rhotermination factor has remained elusive, it has beenwell established that Rho binds preferentially tounstructured 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.10–12

Rho 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 reserve

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 single‐moleculeassay 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 streptavidin‐coated bead and the 5′ end of the nascent RNAtranscript is annealed to a DNA handle attached to aseparate bead, creating a molecular “dumbbell”.This dumbbell is then held on each end using anultra-stable dual optical trap system capable ofapplying a wide range of stretching forces anddetecting 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-calledforce–extension 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 factor–RNA in-

teractions, the authors utilized three different RNA

d.

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662 Getting a Grip on the Terminator

substrates, each possessing the rut site from the λtR1terminator 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, single‐moleculeanalysis of binding footprints does not suffer fromheterogeneity between individual Rho–RNA com-plexes that may obscure results in ensemble foot-printing analyses.Next, the authors exploited the power of their

single‐molecule 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 single‐molecule 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-dependenttermination requires the Rho complex to “catchup” to an actively transcribing RNAP EC, it will beinteresting to study the competing rates of the

RNAP 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 theefficiency of Rho-dependent termination promisesto yield additional insights into the molecularmechanism of transcriptional regulation.

References

1. Peters, J. M., Vangeloff, A. D. & Landick, R. (2011).Bacterial transcription terminators: the RNA 3′-endchronicles. J. Mol. Biol. 412, 793–813.

2. Roberts, J. W. (1969). Termination factor for RNAsynthesis. Nature, 224, 1168–1174.

3. Gogol, E. P., Seifried, S. E. & von Hippel, P. H. (1991).Structure and assembly of the Escherichia coli tran-scription termination factor rho and its interactionwith RNA. I. Cryoelectron microscopic studies. J. Mol.Biol. 221, 1127–1138.

4. Skordalakes, E. & Berger, J. M. (2003). Structure ofthe Rho transcription terminator: mechanism ofmRNA recognition and helicase loading. Cell, 114,135–146.

5. Thomsen, N. D. & Berger, J. M. (2009). Running inreverse: the structural basis for translocation polarityin hexameric helicases. Cell, 139, 523–534.

6. Bogden, C. E., Fass, D., Bergman, N., Nichols, M. D. &Berger, J. M. (1999). The structural basis for terminatorrecognition by the Rho transcription terminationfactor. Mol. Cell, 3, 487–493.

7. Briercheck, D. M., Wood, T. C., Allison, T. J.,Richardson, J. P. & Rule, G. S. (1998). The NMRstructure of the RNA binding domain of E. coli rhofactor suggests possible RNA–protein interactions.Nat. Struct. Biol. 5, 393–399.

8. Skordalakes, E. & Berger, J. M. (2006). Structuralinsights into RNA-dependent ring closure andATPase activation by the Rho termination factor.Cell, 127, 553–564.

9. Lowery-Goldhammer, C. & Richardson, J. P. (1974).An RNA-dependent nucleoside triphosphate phos-phohydrolase (ATPase) associated with rho termina-tion factor. Proc. Natl Acad. Sci. USA, 71, 2003–2007.

10. Chen, C. Y. & Richardson, J. P. (1987). Sequenceelements essential for ρ-dependent transcriptiontermination at λtR1. J. Biol. Chem. 262, 11292–11299.

11. McSwiggen, J. A., Bear, D. G. & von Hippel, P. H.(1988). Interactions of Escherichia coli transcriptiontermination factor rho with RNA. I. Bindingstoichiometries and free energies. J. Mol. Biol. 199,609–622.

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, 19082–19088.

13. Schwartz, A., Margeat, E., Rahmouni, A. R. &Boudvillain, M. (2007). Transcription terminationfactor rho can displace streptavidin from biotinylatedRNA. J. Biol. Chem. 282, 31469–31476.

14. Abbondanzieri, E. A., Greenleaf, W. J., Shaevitz, J. W.,Landick, R. & Block, S. M. (2005). Direct observation

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of base-pair stepping by RNA polymerase. Nature,438, 460–465.

15. Jin, D. J., Burgess, R. R., Richardson, J. P. & Gross, C. A.(1992). Termination efficiency at rho-dependent termi-nators depends on kinetic coupling between RNApolymerase and rho. Proc. Natl Acad. Sci. USA, 89,1453–1457.

Michael D. StoneDepartment of Chemistry and Biochemistry

and the Center for Molecular Biology of RNA,University of California Santa Cruz, Santa Cruz,

CA 95064, USAE-mail address: [email protected]