Protein Science (1992), I , 861-873. Cambridge University Press. Printed in the USA. Copyright 0 1992 The Protein Society

Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. 11. Binding of RNA

JOHANNES GEISELMANN,’ THOMAS D. YAGER,2 AND PETER H. VON HIPPEL Institute of Molecular Biology and Department of Chemistry, Universityof Oregon, Eugene, Oregon 97403 (RECEIVEDDecember 27, 1991; REVISEDMANUSCRIPT RECEIVEDMarch 3, 1992)

Abstract The rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho-dependent termination sites on theDNA template, and (in a series of steps that arestill largely undefined) brings about transcript termination at these sites. In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional form of rho. We use CD spectroscopy and analytical ultracentrifugation to determine the binding interactions of rho with RNA ligands of defined length ([rC], where n 2 6). Rho binds to long RNA chains as a hexamer characterized by D3 symmetry. Each hexamer binds -70 residues of RNA. We show by ultracentrifugation and dynamic laser light scattering that, in the presence of RNA ligands less than 22 nucleotide residues in length, rho changes its quaternary structure and becomes a homogeneous dodecamer. The dodecamer contains six strong binding sites for short RNA ligands: i.e., one site for every two rho protomers. The measured association constant of these short RNAs to rho increases with increasing (rC), length, up to n = 9, suggesting that the binding site of each rho protomer interacts with 9 RNA nucleotide residues. Oligo(rC) ligands bound to thestrong RNA binding sites on the rhododecamer do not significantly stimulate the RNA-dependent ATPase activity of rho. Based on these features of the rho-RNA interaction and other experimentaldata we propose a molecular model of the interaction of rho with its cofactors. Keywords: Escherichiu coli; ligand binding; helicase; quaternary structure; rho; RNA

The Escherichia coli protein rho is required for correct transcript termination at numerous specific (rho-dependent) termination sites on the DNA template (for reviews see PIatt & Bear, 1983; von Hippel et al., 1984; Platt, 1986; Yager & von Hippel, 1987; Richardson, 1990). At other specific (intrinsicor factor-independent) sites, RNA polymerase can terminate transcription without the participation of protein cofactors. Both thermodynamic and & Yager , 1991, 1992; Yager kinetic arguments (von Hippel & von Hippel, 1991) have been proposed to explain termination at such intrinsic sites. A central feature of these

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Reprint requeststo: Peter H. von Hippel, Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403. Present address: Departement de Biologie Moleculaire, Universitt de Geneve, 30, Quai Ernest-Ansermet, CH-1211 Genkve 4, Switzerland. Present address:NSF Center for Molecular Biotechnology, Division of Biology (139-74), California Institute of Technology, Pasadena, California 91 125.

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arguments is that the very stable and processive transcription elongation complex is destabilizedby the formation of a hairpin in the RNA transcript within the “transcription bubble” as the polymerase approaches the factorindependent termination site. The formation of this hairpin and the concomitant disruption of the RNADNA hybrid isthen thought to destabilize the elongation complex, resulting in release of the nascent RNA. No comparably detailed description has yet been proposed for the mechanism ofrho-dependent transcription termination. This process requiresa region of the nascent RNA that is reasonably free of secondary structure and relatively richin cytosine residuesas a loading site where rho initially binds (Morgan etal., 1985; Chen et al., 1986; Alifano et al., 1991). Termination then occurs at specific downstream siteson the DNA template at which the elongation complex undergoes lengthy(and rho-independent) pausing events(Lau et al., 1983; Morgan et al., 1983a,b). The precise mechanism by which rho brings about transcript release at these sites is still unknown.

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The enzymatic properties of rho can be studied in the absence of the transcription complex. Rho carries an RNA-dependent ATPase activity that displays an RNA cofactor requirement similarto that of rho-dependent termination: i.e., a rhobinding site on the RNA that is relatively devoid of stable secondary structure and relatively rich in cytosine residues.This ATPase activityand its cofactor requirements have been extensivelystudied (Lowery & Richardson, 1977a,b; Galluppi & Richardson, 1980; McSwiggen, 1985; von Hippel et al., 1987; Faus & Richardson, 1989). Using this RNA-dependent ATPase as an energy source, Brennan et al. (1987) have shown that rho can act as a 5' 4 3' directional RNA-DNA helicase on RNA-DNA heteroduplex constructs that carry a singlestranded region of RNA at the 5' end. This helicase action is thought to be central to the process of transcript release at rho-dependent termination sites. The interaction of rho with RNA seems to be implicated in all the functions of the protein. In this paper we describe in detail some aspects of the interaction of rho with homopolymer RNAs of varyinglength. Three complementary techniques (CD, analytical ultracentrifugation, and dynamic laser light scattering) have been used to monitor the binding of RNA to rho in this study. We have used these results to characterize some aspects of the binding interactions of rho with RNA, and, in particular, have observed changes inthe state of association and the conformation of rho that result from RNA binding. The combination of these data with experiments on ATP binding (Geiselmann & von Hippel, 1992) and with published properties of the rho-RNA system allows usto propose a physical model for how rho might catalyze transcript termination (Geiselmann et al., in prep.).

contributions by including a control cell in the rotor that contains only rho at the same concentration as in the experimental cell. In this way we have been able to quantitate the concentration of bound and free RNA as a function of position within the ultracentrifuge cell (see Revzin & von Hippel, 1977). The CD approach makes use of the fact that oligomers of cytosine have a strong positive CD signal at 275 nm. When these ligands are bound to rhotheir conformation changes, resulting in an -50% decrease in the molar ellipticity of the RNA at this wavelength. The conformational change induced by the binding of RNAto rho may be very subtle, and binding modes that do not change the conformation of the RNA ligand are, of course, not detected by this technique. Therefore we use this spectroscopic approach in conjunction with analytical ultracentrifugation, which detects RNA binding directly by changes in the profile of the sedimentation boundary. Wewish to determine if RNA binding is coupled to changes in the quaternary structure of rho. It is therefore also necessary to measure changes in association state of rho (in the absence of RNA ligand) under the conditions of the binding experiments. We have used analytical ultracentrifugation and dynamic laser light scattering to obtain this information. We first present the ultracentrifugation data in order to define the quaternary structure of rho when complexed to RNA ligands of various length. These experiments also yield information about the stoichiometry of RNA binding. The binding interaction is then analyzed in detail using the CD titration method. The physiological association state of rho

Results We have studied the interaction of rho with model RNA cofactors, using defined-length oligomers of cytosine as binding ligands. The use of homo-oligonucleotide cofactors eliminates sequenceeffects and the formation of secondary structure that may complicate experiments with natural RNAs. Oligo(rC) ligands were chosen for these studies because cytosine-containing oligo- and polynucleotides maximally stimulate the ATPase activity of rho (Lowery & Richardson, 1977b). We have monitored RNA binding to rho by analytical ultracentrifugation and CD spectroscopy. Short RNA ligands do not sediment appreciably during ultracentrifugation when they are not bound to rho (see Fig. 1). RNA ligands that are bound to rhosediment with the protein. The relative heights ofthe nonsedimenting and sedimenting boundaries in the ultracentrifuge cell reflect the ratio of the absorbance of the free RNA to that of the rho-RNA complex. The absorbance of the rhoRNA complex is separated into protein andnucleic acid

Rho binds (rC),o as a hexamer We have previously described conditions under which a stable, homogeneous hexamer of rho exists in solution (Geiselmann et al., 1992b). This hexameric form of rho is thought to represent the physiologically relevant association state and appears to be the form that binds to long RNA chains in solution (Bear et al., 1988; Gogol et al., 1991). Also, the rho hexamer, when bound to long RNA, displays the ATPase activity that is required for rho& dependenttranscriptiontermination(Richardson Conaway, 1980; McSwiggen et al., 1988). Based on a number of physical studies, we have proposed a model for the structure, symmetry, and assembly of the rho hexamer (Seifriedet al., 1991; Geiselmann et al., 1992a,b). In this model six identical rho protomers are arranged as a planar hexagon characterized by D3 symmetry. As the total concentrationof rho is lowered, the rho hexamer dissociates (Finger & Richardson, 1982; Seifried et al., 1991); the addition of poly(rC) prevents this dissociation. Finger and Richardson (1982) observed a rho-poly(rC) complex that sediments at 12s in a glyc-

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863

RNA binding to rho protein

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top of cell

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12

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18

18

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t bottom of cell

Fig. 1. Velocity sedimentationprofile of a mixture of rhoand ~ligo(rC)~. The absorbance at 280 nm is shown as a function of the distance from the top of the centrifuge cell. Rho (8.0 p M protomers) contributes 0.12 OD to the total absorbance, and (rC), (9.6 p M in ligand molecules) contributes 0.46 OD. The absorbance within the sedimenting boundary is due to rho and toRNA bound to rho. The contribution of rho can be determined independently by comparison with a cell, spun within the same rotor, containing only rho protein at the Same concentration. The remaining absorbance within the sedimenting boundary is attributed to bound RNA and corresponds to a concentration of The free RNA (-6 p M ) does not sediment. With a 3.5-4 p M of limiting stoichiometry of three RNA molecules bound per rho hexamer, this corresponds to a binding saturation of 85-100%, which translates into a binding constant >lo6 M" .

erol gradient. We have confirmed this finding and extended it to higher concentrations of rho. An equimolar mixture of (rC)70 and rho hexamer is sedimented in the analytical ultracentrifuge. In Figure 2 we plot the integral distribution of the sedimentation coefficient for a number of different rho-RNA complexes (see van Holde & Weischet [ 19781 for details of this type of analysis of a sedimentation boundary). The data for rho alone (which exists as a hexamer under these conditions [see Geiselmann et al., 1992b]), and for rho bound to (rC)70at a stoichiometry of one ligand per rho hexamer, are shown in the top panel of Figure 2. The rhoRNA complex sediments faster than the unliganded rho hexamer, but considerably more slowly than does the rho dodecamer described below. (The extra mass of the bound RNA increases the observed sedimentation coefficient by only about 5 % , assuming no major conformational change occurs in the rho hexamer.) The rh~-(rC)~,, complex appears to be somewhat heterogeneous, as indicated by the sloped plot for this species. This may reflect some concentration-dependent aggregation due to the formation of bridged complexes in which single RNA molecules are bound to different rho hexamers (as seen with rho-[rCId5 complexes described below). It could also reflect a general affinity of rho-RNA complexes for one another, which would be consistent with the observation that hexamers bind cooperatively to poly(rC) (Bear et al., 1988; McSwiggenet al., 1988). We note that the hexameric limit is attained at the low concentration end of the sedimentation boundary in our experiments.

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22201816 8 141210

8 (Svedberg) Fig. 2. Velocity sedimentation of rho and RNA. The sedimentation boundary of rho and RNA in a model E ultracentrifuge is analyzed according to van Holde and Weischet (1978).A sedimentation boundary (e.g., Fig. 1) is divided into 14 equispaced levels; these levels are plotted along the ordinate. On theabscissa we plot the corrected sedimentation coefficient, szo,,,., by extrapolating that level of the boundary to infinite time. A vertical line means that each part of the sedimenting boundary extrapolates to the same s20, at infinite time. Such behavior is typical of a homogeneous sample (and of heterogeneous species in rapid equilibrium). A gradual change in szO, along the boundary indicates sample heterogeneity. In the presence of nonsedimenting material (e.g., free RNA ligand) the analysis is applied only to the sedimenting part of the boundary (see textand above). The sedimentation coefficients characteristic of the rho hexamer and dodecamer are indicated as shaded areas. All samples were run in 0.1 M KCl, unless otherwise indicated. The concentration of rho is in p M protomers, and the concentration of RNA ligands is in p M molecules. Top panel: (a) rho at 13.8 pM (filled circles); (b) rho at 6.6 p M plus 1.3 p M (rC)70(filled triangles); (c) rho at 13.8 p M plus 3.2 p M (rC)70 (open circles); (d) rho at 4.1 p M plus ~ ~ squares); (e) rho at 4.1 p M plus 1.5 p M ( T C ) ~ ~ 0.74 p M ( T C ) (open (open triangles); (f) rho at 16.8 p M plus 9.0 pM (rQI3 (filled squares). Bottom panel: (a) rho at 12.0 p M plus 2.0 p M (dC),o (filled triangles); (b) rho at18.6 p M in 0.5 M KC1 (open circles); (c) rho at 6.6 p M plus 3.7 p M (rQ1 in 0.56 M KC1 (filled circles); (d) rho at 6.6 pM plus 3.7 pM (rQl1 in 0.1 M KC1 (filled squares).

Other association states of rho A complex between the rho hexamer and an RNA chain that is several hundred nucleotide residues long probably is most representative of the situation in vivo. However, much can also be learned by studying association states generated under less physiological conditions. For example, the validity of the D, model of the rho hexamer can be tested, and information about the nature of the interaction of rho with its RNA ligand can be obtained. The properties thus revealed may have implications for the function of rho under physiological conditions. In this section we describe other association states of rho and specify the conditions under which they are generated. In subsequent sections we examine one of these states in de-

864 tail to learn more about the nature ofthe binding interaction between rho and the RNA ligand. Rho complexed with short RNA ligands Rho is converted from a hexamer into a homogeneous dodecamer in the presence of RNA ligands that are 9-20 nucleotide residues in length. Some of the data that document this point are shown in Figure 2. In these experiments rho was sedimented in the presence of cytidine oligonucleotide ligands that were between 9 and 20 residues long. Rho concentrations ranged from 4 to 20 pM (in promoters). The free oligonucleotides, which do not sediment under the conditions of the experiment (see Fig. I), were at concentrations of 1-10 pM (in ligand molecules). The rho-RNA complexes sediment as homogeneous species with a corrected sedimentation coefficient (S20,w)of 17.5-18s. Dynamic laser light-scattering experiments with this material yield a corrected diffusion coefficient (Dzo,), of 2.8 x 10” cm2/sec. Combination of the S and D values of a homogeneous species allowscalculation of its molecular weight via the Svedberg equation and confirms that rho forms a dodecamer under these conditions (Geiselmann et al., 1992b). The dodecameric form of rho is generated by binding oligo(rC) (or oligo(dC)) ligands that are 9-20 nucleotide residues in length (see also Fig. 2). In Geiselmann et al. (1 992b) we presented sedimentation velocity, quasielastic light-scattering, sedimentation equilibrium, small angle X-ray scattering, and small angle neutron scattering data that show unambiguouslythat the dodecameric state is homogeneous and well defined. The dodecamer appears to consist of two planar hexagons stacked on top of each other (see also Gogol et al., 1991). Most of theseexperiments were conducted with oligo(rC), although several experiments were performed with oligo(dC) as well. We find that (dC)Io and (dC),, show the same binding behavior as their oligo(rC) counterparts, suggesting that the binding sites on rho that are probed in these experiments have the same affinity for both types of nucleic acid oligomers. Thisis in agreement with earlier binding titration studies with poly(rC) and poly(dC) (McSwiggen et al., 1988).

Rho complexed with RNA ligands of intermediate length Interaction of rho with polymeric RNA leads to a predominantly hexameric association state. In contrast, interaction of rho with short RNA oligomers results in the formation of a highly homogeneousdodecamer. We have studied the binding to rho of one RNA ligand of intermediate length (45 nucleotide residues). In these experiments a binding stoichiometry of either one or two RNA ligands per rho hexamer was used and a heterogeneous distribution of products, all larger than dodecamers, was obtained (Fig. 2, top panel). All of the RNA was bound

J. Geiselmann et al. at both stoichiometries examined. In the Discussion we attempt to rationalize the peculiar behavior of this intermediate-size RNA on the basis of the binding stoichiometries observed with RNA ligands of different lengths. Binding interactions of rho with RNA

Having established the quaternary structure of the different rho-RNA complexes, we now address in detail the binding of RNA to rho. One goal of these studies is to understand the physical interaction between a rho protomer and its RNA ligand. A second goal, which may be hard to separate from the first, is to understand the cooperativity of ligand binding, i.e., how RNA binding to one rho protomer within a rho hexamer (or dodecamer) may affect the binding of subsequent RNA ligands. A comparison of the binding of long and short RNAs allows us to begin to address these questions as a short RNA oligonucleotide can only span the binding site of a single rho protomer, whereas a longer oligonucleotide can make simultaneous contact with several binding sites on the same hexameror even form a bridge betweenrho hexarners (see the Discussion). RNA binding monitored by CD spectroscopy

The sedimentation velocity experiments described above show that the binding of short RNA ligands to rhoresults in the formation of a homogeneous dodecamer of rho. These experimentsalso show that other association states of rho are generated on binding longer RNAs.In this section we examine these bindinginteractions in detail, using quantitative CD titration to determine equilibrium binding constants and binding stoichiometries. Figure 3 shows the results of experiments in which the rho hexamer is titrated with (rc)6, (rC)8, or ( r Q 3 . The (rc), ligand shows weak binding with an estimated equilibrium constant of less than 0.2 X IO6 M-’. A precise binding constant cannot be extracted from these data because of the very small extentof binding that is observed. However a simulated titration experiment indicates that a binding constant of 0.2 x lo6 M” can be reliably determined by our curve-fitting routine. In contrast, the longer oligo(rC) ligands bind to rho with significantly larger equilibrium constants and show defined titration endpoints. The next larger RNA ligand investigated, (rC)s, shows a binding constant of 4 x lo6 M” . (All binding constants reported are per mole of ligand nucleotide residue and per mole of rho protomer.) This titration is almost stoichiometric, and the fit of the site size and the binding constant to the data arereliable; 95% confidence in1. tervals for these parametersare showninTable Titration with (rC)13 shows very tight binding of this ligand to rho(Fig. 3). For RNA ligands of this length the exact value of the binding constant cannot be reliably de-

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RNA binding to rho protein 5 4 P,

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[nucleotides] in pM Fig. 3. CD titration of rho with oligo(rC) ligands of different length. The rhoconcentration is 2 pM. TheCD signal due to the RNA oligomer alone has been determined in a parallel titration and the straight line fit subtracted from thedata presented. The solid curves represent the best fit to the data using Equation 1. The topcurve (triangles) monitors the binding of ( r Q 3 , the middle curve (filled squares) monitors (rC), binding, and the bottom curve (open squares) monitors (rc), binding. All binding curves have been corrected for the volume increase upon ligand addition.

Table 1. CD titration of rho with oligo(rC)a Length 6 8 8 (ATP) 9 12 12 (ATP) 13 22 46 72 130 Poly(rC)

[Rho] in pM protomers

Site size (n) (95% confidence)

3.00 3.00 3 .oo 2.00 1.60 1.60 2.00 0.50 0.93 0.93 1S O 1.80

- 100.0 (68, 100) 57.0 (20, 100) 8.0

"All titrations were carried out in the standard binding buffer: 40 mM Tris-HC1 (pH 7.8), 100 mM KCI, 10 mM MgClz, 0.1 mM EDTA. A 1.8-mL volume was titrated by adding aliquots of RNA oligomer (-300 pM nucleotides). The titration was continued up to a final RNA concentration of -5x its value at the equivalence point (typically -50 pM). The binding curves were fit to Equation 1 to obtain asite size and a binding constant (expressed in moles of RNA nucleotide residues and moles of rho protomers). The apparent binding constant for poly(rC) with Equation 1 is too low. Using the appropriate equation incorporating overlap and cooperativity (see text) we obtain a binding constant ( K w ) of 3 X lo8 M-l for this ligand. Upper (95%) confidence interval limits of 100 indicate that the confidence limit has not been reached at this value; i.e., a very good fit could be obtained with even higher binding constants.

termined; however, we can show that this binding constant is on the order of 50 x lo6 M" . We conclude that oligomers longer than 9 nucleotide residues are very strongly bound by rho. Differences in binding affinities within this class of very strongly binding oligomers cannot be reliably established by these techniques. The results of many such titrations are summarized in Table 1. We show that for some of these ligandsthe same values of K and n are obtainedwhen the titration is performed in the reverse direction (see Table 2). In some of the titration experiments we have also included the nonhydrolyzable ATP analogue, AMP-PCP, at a concentration of 20 pM, whichiswell above the concentration needed for appreciable binding to rho (Stitt, 1988). The binding of the RNA ligands is not altered by the presence of this compound (see Tables 1 and 2). An interesting anomaly appears in Tables 1 and 2, in that the apparent binding site size ( n ) seems to increase with the length of the RNA ligand used in the titration. The values for this parameter range from n = 5 for short RNA oligomers (8-13 residues in length) to n = 11 for RNA oligomers longerthan 45 residues. We interpret this finding in terms of a model in which strong and weak RNA binding sites alternate on the rho hexamer or dodecamer. Long RNA molecules can span the distance between two strong binding sites on the same hexamer, whereas sfiort RNAs cannot. At the RNA concentrations we have used in this study only polymeric RNA will bind to the weak sites. This latter binding may simply be a consequence of polymer connectivity, as the parts of the polymer bound to the strong sites should serve to bring intervening parts of the same polymer close to the weak sites on the rho hexamer. In experiments currently in progress we are examining the binding properties of the weak sites at very high concentrations of short RNA li-

Table 2. CD titration of oligo(rC) with rho a Length

6 5.112 12 (ATP) 13 22 46 72 Poly(rC)

[Oligomer] in pM nucleotides

17.0 11.0 13.6 11.2 19.0 13.0 28.5 26.0 19.6

Site size (n) -

5.3 4.8 14.3 11.3 11.4 11.2

K ( x lo-, M-I)