Proc. Nati. Acad. Sci. USA Vol. 91, pp. 1401-1405, February 1994 Biochemistry Evidence supporting a tethered tracking model for activity of Rho factor ( termination/translocation/ATPase/RNA- interactions/hexameric helicase) ERic J. STEINMETZ* AND TERRY PLATTt Department of Biochemistry, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642 Communicated by Donald M. Crothers, October 22, 1993 (receivedfor review April 15, 1993)

ABSTRACT Transcription Rho of observation that Rho may remain associated with its initial Escherichia coli has an ATP-dependent RNADNA helicase site while continuing ATP hydrolysis (12). In this "tethered activity that presumably facilitates RNA transcript release tracking" model, Rho maintains its primary RNA binding from the elongation complex. This helicase activity is unidi- interaction with trp t' while interactions at secondary RNA rectional (5' to 3') and is stoichiometric, with one RNA sites, coupled to ATP hydrolysis, extend Rho's contacts molecule released per Rho hexamer in vitro. A simple RNA along the RNA. tracking model postulates that after Rho's initial binding, it We have tested these models by measuring the dissociation translocates preferentially toward the 3' end of the RNA. rates of Rho-RNA complexes under helicase reaction con- Nitrocellulose filter binding studies combined with RNase H ditions and then combined filter binding with targeted RNase cleavage are inconsistent with this simple tracking model. H digestion to examine the position of Rho on long RNA Instead, they support a model in which Rho forms tight molecules after it has carried out helicase activity at different primary binding interactions with the recognition region of the sites. Our results favor a tethered tracking mechanism for RNA and remains bound there while transient secondary RNA Rho in transcription termination. binding interactions coupled to ATP hydrolysis serve to scan along the RNA to contact the RNADNA helix. This "tethered MATERIALS AND METHODS tracking" model is consistent with other properties of Rho factor, including the presence of two classes of RNA binding Rho Protein. Rho protein was purified from the E. coli sites on the Rho hexamer and the 1:1 stoichiometry in the Rho strain AR120-A6 containing a Rho-overproducing plasmid as helicase assay. previously described (13), except that plasmid p39-ASE, containing a corrected version of the rho gene, was used (14). The original plasmid contained a mutation at codon 155, Escherichia coli Rho factor mediates ATP-dependent RNA resulting in a lysine substitution for glutamic acid (EK155). release from RNA polymerase elongation complexes at Rho- Comparison of the activities of the wild-type and EK155 dependent transcription termination sites (1-3). ATP hydro- revealed little or no difference in RNA binding, lysis by Rho is RNA-dependent, potentiated by specific RNA RNA-DNA helicase, and transcription termination activities, sequences upstream of the 3' endpoints specified by Rho and a 2-fold difference in Vmax of ATP hydrolysis (14). Rho action (4, 5), and is thought to fuel motion by Rho protein concentration was determined as described (13) and is ex- toward the transcription elongation complex. Rho's 5'-to-3' pressed as the hexameric form of the protein. RNADNA helicase activity (6) probably facilitates termina- Nucleic Acids. 32P-labeled RNA was synthesized in vitro tion by disrupting the RNA-DNA hybrid in the transcription from SP6 templates with [a-32P]GTP as labeled substrate "bubble." In particular, RNA containing the Rho-dependent (6-8). RNA-RNA duplexes were formed in hybrid formation termination site trp t' directs the efficient disruption of buffer (150 mM KCl/20 mM Tris-HOAc, pH 7.9/0.1 mM downstream RNADNA helices in vitro (6-8), even when EDTA) (7, 8). The A cro template was provided by J. P. nonfunctional sequences are interposed between the two Richardson (Indiana University). Oligodeoxynucleotide d(G- regions (8). An intervening RNARNA hybrid structure acts CATCGTCGATACCC) was obtained from Oligos Etc. (Guil- as a conditional block to disruption of the downstream ford, CT). RNA-DNA helix, suggesting that Rho must track along the RNA Binding Assays. A nitrocellulose filter assay for RNA RNA (8). Maximal RNA release is achieved within about 1 binding (9, 15) was used for the dissociation rate experiments: min, yet only one RNA molecule is released per Rho hexamer 1 nM Rho was preincubated with 1 nM 32P-labeled RNA at even after long incubation times (6-8). ATP hydrolysis 370C for 2-3 min in helicase reaction buffer (50 mM KCI/20 continues long after maximal release, indicating that Rho mM Tris-HOAc, pH 7.9/1 mM Mg(OAc)2/1 mM ATP/0.1 remains productively bound to RNA (7), an interpretation mM EDTA) in 145 tkl before addition of 5 A.l of competitor supported by studies of a mutant Rho protein that can RNA (or water) to 0, 10, 20, or 30 nM. Aliquots (20 IL) were catalyze multiple rounds ofRNA release in the helicase assay removed at 3-min intervals for filtration. Filters were sub- by virtue of a 160-fold weaker affinity for trp t' RNA (9). jected to liquid scintillation counting to determine the percent Any mechanism for 5'-to-3' action at a distance must RNA bound relative to the zero time point, which was accommodate these findings. A simple tracking model in- defined as 100% (the initial percentage oflabeled RNA bound vokes 5'-to-3' translocation, such that after binding to the trp was 30-50% for HT, 50-70% for T', and 55-70% for A cro t' region, Rho then moves along the RNA, leaving its initial RNA). binding site and ending up near the RNA 3' end. An alter- For experiments combining RNase H cleavage and filter native model is based in part on a two-site model for RNA binding, reaction mixtures contained 4 mM Mg(OAc)2 and 5 binding and ATPase activation (10, 11), as well as the mM ATP to allow efficient disruption of RNARNA helices

The publication costs of this article were defrayed in part by page charge *Present address: Department of Biomolecular Chemistry, Univer- payment. This article must therefore be hereby marked "advertisement" sity of Wisconsin, 1300 University Avenue, Madison, WI 53706. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 1401 Downloaded by guest on September 24, 2021 1402 Biochemistry: Steinmetz and Platt Proc. Nati. Acad. Sci. USA 91 (1994)

(7, 8). After filtration ofthe samples, filters were washed with pM (data not shown), in agreement with published values (9), 75 gl of reaction bufer, and both the flowthrough and wash suggesting that Rho might dissociate very slowly from trp t' were collected. RNA bound to the filter was eluted slowly RNA. To measure the dissociation rate directly, we used a with two 15-4 aliquots of 0.5 M NaCl typically yielding competition assay in which excess unlabeled trp t' RNA was 95-98% of the RNA. Samples of bound and free RNA were added to preformed complexes ofRho and labeled RNA, and adjusted to 0.25 M NaCl in 15 gl before addition of 15 ul of aliquots were filtered through nitrocelhlose at intervals over 80% formamide/0.1% bromphenol blue/0.1% xylene cyanol. a time course of 12 min. The decrease in bound radioactivity Sample$ were heated briefly to 950C before electrophorrdsis in has three components: true dissociation of Rho-RNA com- a 5% polyacrylamide/7 M urea gel in TBE buffer (50mM Tris plexes, inactivation of Rho protein, and displacement of borate, pH 8.3/1 mM EDTA). Individual bands localized by labeled RNA by the unlabeled competitor at a rate that is autoradiography were excised from the gel for liquid scintil- dependent on the competitor concentration (11). These pseu- lation Counting. Alternatively, for the experiments in Fig. 3, do-first-order kinetics are described in Fig. 1 and Table 1. autoradiograph bands were quantitated by laser densitometry At 10 nM competitor RNA, preformed complexes of 1 nM with ad LKB Ultroscan densitometer to allow correction for Rho and 1 nM trp t' RNA decayed with ka,,p = 0.07 min' (ti/2 background. Band intensities were determined by cutting and 10 min). The major contributors to this overall decay rate weighing peaks from the densitometer plot. Because the are the displacement (k2,disp = 0.0026 minl-inM-1) and inac- RNA is internally labeled with [a-32P]GTP and the trp t' tivation (kinc = 0.027 min') reactions; the remaining value regio$ is relatively poor in G residues, the smaller UT attributable to dissociation is 0.018 min' (t1/2 39 min). This fragment has 1.7 times more label than the T' fiagment, and accounts for the 1:1 stoichiometry of Rho in the helicase full-length T'HT RNA is 2.9 times as radioactive. To com- assay; at these low concentrations (1 nM Rho hexamers = pensate for any loading errors, only bands from the same lane 0.28 pg/ml), Rho inactivates faster than it dissociates from were compared directly. RNA. Fig. 1B and Table 1 also present data from the decay of complexes with either the A cro mRNA, containing the tRi RESULTS Rho-dependent termination region (15), or a nonspecific Rho Dissociates Slowly from RNA Under Helicase Reaction RNA, HT, ofthe same size as trp t' but a very poor activator Conditions. To measure the affinity of Rho for trp t' RNA in of RNA-dependent ATPase and helicase activities (8). Both helicase reaction buffer, Rho protein was titrated against a RNAs dissociated slowly from Rho, with half-lives of 26 and fixed concentration of RNA and the amount of RNA bound 7 min, respectively. to Rho was determined by a nitrocellulose filter assay (9, 15). Rho Is Preferentiaily Retained at op t' After Ease Action Results indicated a very small dissociation constant (Kd), =10 at a Distance. The slow dissociation ofRho-RNA complexes, the 1:1 stoichiometry of helicase activity, and the continued 0.2 hydrolysis of ATP are consistent with Rho remaining bound to RNA after carrying out helicase activity. To examine what 0.0 portion of an RNA molecule Rho is bound to after disrupting o -0.2 a downstream helix, a chimeric RNA, T'HT, containing the 0 trp t' Rho-dependent termination region upstream of a non- - -0.4 specific spacer sequence (HT), was annealed to a short complementary RNA (THA19), forming a 50-bp RNARNA a:-0.6 duplex near the junction between the trp t' and HT segments I.-, -0. 8 (Fig. 2A). A 15-mer DNA oligonucleotide, also complemen- tary within this region and present in 1000-fold excess, will -1.0

-1.2 Table 1. Summary of decay rates for Rho-RNA complexes ti/2, k2,disp, Kd, Time, min RNA kdi~s, min- mmn kinj, min1 min' nM-1 pM trp t' 0.018 ± 0.005 39 0.027 ± 0.004 0.0026 ± 0.0007 10 HT 0.097 ± 0.045 7 0.029 ± 0.006 0.013 + 0.004 40 tR1 0.027 25 0.034 0.0058 20* The apparent decay rate for Rho-RNA complexes at a given concentration of competitor is the negative slope of a plot of In(% RNA bound) versus time (Fig. 1A) and is the sum of these compo- a. nents: kapp = kin + kdiss + (k2,disp X [competitor]). The true -p dissociation rate constant (kdis) is extracted by plotting the apparent dissociation rate versus the competitor concentration and extrapo- lating the resulting straight line to 0 competitor, where ka,(0 com- petitor) = kj,,c + kdiss (Fig. iB). The inactivation rate constant (kijaj) is determined from the rate of decay of Rho-RNA complexes in the absence ofcompetitor (upper line in Fig. 1A), and subtraction ofthis 20 30 value from the y intercept of Fig. 1B yields kdiss. The second-order Competitor, nM displacement rate constant (k2,disp) is equal to the slope of this secondary plot. FIG. 1. Decay of Rho-RNA complexes. (A) Example of a single *Previous studies of Rho-cro interactions found a Kd of 1.25 nM in experiment showing the decay ofpreformed complexes of 1 nM Rho 50 mM KCl/40 mM Tris HCl, pH 8.0/10 mM MgCl2/0.1 mM EDTA and 1 nM 32P-labeled trp t' RNA over time with 0 nM (0), nM(s), (15). We have found that the affmnity depends strongly on the total 20 nM (o), or 30 nM (i) unlabeled trp t' competitor RNA. (B) Plots Cl- concentration, since substitution of10mM Mg(OAc)2 forMgCI2 of apparent d&cay rate versus competitor concentratibn for Rho- or reduction of MgC2 to 1 mM restored the affinity to nearly that RNA complexes. o,' Rho-trp t' RNA (averages and standard devi- in helicase reaction buffer. Helicase activity is inefficient in 10 mM ations frOm io experiments); *, Rho-HT RNA (averages and stan- MgCl2 and efficient but limited to one RNA per hexamer with either dard deviations fropn 7 experiments); *, Rho-A cro RNA (results trp t' or cro RNA in 1 mM MgCl2 or Mg(OAc)2 (unpublished from a single experiment). results). Downloaded by guest on September 24, 2021 Biochemistry: Steinmetz and Platt Proc. Natl. Acad. Sci. USA 91 (1994) 1403 A + ATP by Rho (lane 3), and most ofthe HT fragment flowed through RNA P RNA oligo (lane 4). In this example, 3.8 times more T' fragment than HT trp t' HT fragment was retained on the filter; the average molar ratio from four experiments was 3.6 ± 0.4. As expected, the T'HT Helicase activity RNA was largely protected from digestion by RNase H in the _ nM RNA oligo absence of Rho, with <20% cleavage in a 3' incubation with Rho buffer -b-- -*I- AO. storage (lane 1), whereas incubation with Rho RNA f P , factor resulted in cleavage of >60% of the T'HT RNA (lane trp t HT 2). Control experiments revealed that RNase H was unable to retain RNA on the filters (data not shown). RNase H cleavage 1J DNA oligo Formally, preferential retention of T' could be due to Rho unwinding the RNARNA duplex ahead of itself, allowing rapid DNA annealing and RNase H cleavage before leaving RNA the T' region behind. To test this, we delayed adding RNase trp t' (260 nt) H and monitored the disruption of an RNA*DNA duplex at ? HT (200 nt) the 3' end of the T'HT RNA. Here, too, the T' fragment was preferentially retained, even though Rho had carried out helicase activity 170 nucleotides downstream of the RNase H cleavage site (data not shown). Filter-binding assay Rho Binding to T' Is Not Due to Intermolecular Reassocia- tion. The above results might be due to rapid dissociation of Rho from the HT segment after helicase activity, followed by B 1 2 3 4 5 6 7 8 preferential reassociation with the T' fragment. We therefore repeated the experiment of Fig. 2A with the addition of THT 475 _ * _ 10-fold excess unlabeled trp t' RNA after preincubating the ..~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.. labeled substrate with Rho and before adding ATP. Again, T' 260-- 4* Aw A most of the T' fragment was retained on the filter (Fig. 2B, lane 6), and most of the HT fragment flowed through (lane HT 200-B- * *o 7). If Rho had dissociated from the RNA after helicase activity, most would have rebound to the unlabeled T' competitor RNA. The amount ofRNA cleaved (40%, lane 5) and the total RNA bound in this experiment are slightly reduced in part because of the displacement reaction. Adding the competitor RNA prior to incubation with Rho and ATP reduced RNase THAI9 60 _-- '40 'A' H cleavage to levels seen in the absence of Rho (<20%, lane 8). In the above experiments, some of the HT fragment was FIG. 2. Preferential retention of Rho factor at trp t' after helicase consistently retained on the filters. The competition studies activity downstream. (A) Schematic diagram. Rho is added to ruled out the possibility that Rho moved from T' to HT and preformed RNA-RNA hybrids in the presence of RNase H and 1 .M oligodeoxynucleotide (DNA oligo). Disruption of the RNA*RNA then rapidly dissociated and redistributed among the frag- duplex allows annealing of the DNA oligo, targeting the RNA for ments, but they did not address the possibility of intramo- cleavage by RNase H. (B) Autoradiograph of a gel. Lanes 1-4: Rho lecular redistribution by some rapid bidirectional tracking or was added at a final concentration of 1 nM to helicase reaction buffer intrastrand transfer mechanism after helicase activity and containing 1 nM T'HT THA19 RNA-RNA duplex, 2 units of RNase before RNase H cleavage. In this case, the preferential H (United States Biochemical), and 1 ILM DNA oligonucleotide. retention at T' might simply reflect its higher affinity for Rho. After 3 min at 370C, an aliquot was filtered through nitrocellulose, Alternatively, Rho could remain tightly bound ("teth- and samples of bound and free RNA were electrophoresed (see ered") to the T' segment of RNA throughout the course of Materials and Methods). Lane 1, incubation at 370C with Rho storage helix disruption downstream. If so, the low level of filter- buffer (14) only; lane 2, total reaction (unfiltered) after incubation HT RNA with 1 nM Rho for 3 min at 370C; lane 3, sample trapped on the filter bound fragment might result from two Rho hex- by Rho and then eluted; lane 4, filter flowthrough sample; lanes 5-7, amers binding simultaneously to the T' and HT segments of Rho was preincubated with the labeled RNA-RNA duplex for 1 min the same intact T'HT molecule. Helicase activity by the at 370C without ATP to allow binding, unlabeled competitor RNA upstream Rho molecule would lead to RNase H digestion, was then added to 10 nM, the reaction was started by adding ATP to and both RNA fragments would be retained on the filter. With 5 mM, and the mixture was incubated for 3 min more [lane 5, total the 1:1 ratio of Rho hexamer to RNA substrate used in Fig. reaction (unfiltered); lane 6, sample trapped on filter; lane 7, 2, the probability of multiple binding is significant: 1 nM Rho flowthrough sample]; lane 8, unlabeled competitor and labeled added to a mixture of 1 nM each of precleaved T' and HT substrate were added simultaneously and incubated with Rho and ATP for 3 min. fragments distributed with a 2.5 ± 0.5 ratio of T' to HT bound after a 3-min incubation (data not shown). If Rho associates target T'HT for cleavage by RNase H only if the RNARNA independently with the two segments when they are con- hybrid is first disrupted by nected, this calculation predicts a substantial contribution to R4o bound initially to the T' HT bind4ng by a second Rho hexamer even when binding by region. The resulting T' and HT fragments were examined by a first Rho molecure at T' results in helicase activity and nitrocellulose filter binding, to separate free RNA from that RNase H cleavagej. bound to Rho. Persistent Assoei'ation with T' Is Not Due to Intramolecular In a simple unidirectional tracking model, Rho should have Redistribution. To distinguish whether rapid intramolecular migrated from the T' segment to the HT segment of the RNA redistribution from HT to T' accounts for the reduced pro- after disruption of the RNA-RNA duplex and should have portion of HT fragment bound after helicase activity, we accumulated there, given the slow dissociation rates pre- repeated the experiment of Fig. 2A at the much higher sented above. However, the opposite was observed (Fig. substrate/Rho ratio of 5:1. The probability of multiple hex- 2B): the T' fragment was preferentially retained on the filter amers binding a single RNA is thus diminished, while intra- Downloaded by guest on September 24, 2021 1404 Biochemistry: Steinmetz and Platt Proc. Natl. Acad. Sci. USA 91 (1994) molecular redistribution events should be unaffected by for at most a 3- to 4-fold preference for binding to T'. substrate concentration. Most of the T'HT RNA now re- Furthermore, the similar ratios observed on initially intact or mains protected from cleavage by RNase H, since Rho is precleaved RNA suggest that intramolecular events do not substoichiometric. With 5 nM RNA-RNA duplex substrate contribute significantly to net changes in distribution. and 1 nM Rho (Fig. 3A), the T' fragment was preferentially retained to a much greater extent than in Fig. 2B, with 13.3 times more T' than HT fragment bound (average from three DISCUSSION experiments). This suggests that the higher proportions ofHT A striking characteristic of Rho-dependent transcription ter- fragment bound in Fig. 2B can be attributed to a contribution mination is the requirement for a specific binding or "entry" from substrates with two Rho molecules bound at once, site on Rho's RNA substrate, and in this aspect Rho differs rather than to intramolecular redistribution. from many other . This binding is a prerequisite for Several other experiments at the elevated substrate con- the activation of Rho to hydrolyze ATP and carry out its centration reinforce this interpretation (Fig. 3B). In each subsequent helicase or transcription termination function. experiment, filter binding reveals Rho's overall distribution Rho's interactions with its natural sites are remarkably among the T' and HT segments, rather than only those Rho strong, with a dissociation constant of 10 pM (tl12 > 30 min) molecules that have been activated by initial binding to the T' for trp t', several orders of magnitude tighter than to random region to unwind an RNA-RNA duplex. In the first experi- sequences of similar size (16). Once bound to RNA, Rho is ment, Rho was incubated with intact, unhybridized T'HT thus temporally tethered to its ultimate target, the RNA-DNA RNA in the presence of ATP and RNase H but without the duplex between the nascent transcript and DNA template in oligodeoxynucleotide for 2 min, allowing time for any intra- the elongation bubble. Previous work showed that such molecular redistribution before addition of the oligodeoxy- tethering alone is not sufficient for helicase function via a nucleotide for 1 min. Since the rate of oligodeoxynucleotide "looping" mechanism and that, instead, Rho must form annealing under these conditions is fairly slow, only about contacts with the intervening RNA via some "tracking" 50% ofthe input RNA was cleaved in this case. In the second mechanism for helicase action at downstream sites (8). experiment, Rho was incubated for 2 min with intact T'HT We have now addressed whether the tracking itself is RNA that was prehybridized to the oligodeoxynucleotide, "linear" or "tethered." Our results, based on the location of and RNase H was added for 1 min. In the third control, Rho's Rho on radiolabeled RNA substrates upon which it has distribution was measured after a 3-min incubation with carried out 3' duplex disruption after initial binding to the 5' precleaved T' and HT fragments, with no possibility for region, rule out unidirectional linear tracking and provide intramolecular redistribution. support for a tethering model. Under a variety of conditions For each control experiment, significantly higher propor- the trp t' RNA is preferentially retained by Rho on nitrocel- tions of the HT fragment were retained on the filter than in lulose filters, compared with the distal HT RNA fragment, the RNA-RNA duplex experiment in Fig. 3A. The observed whereas the clear prediction of a model invoking unidirec- molar ratios of T' to HT fragments bound were 4.4, 2.5, and tional 5'-to-3' locomotion ofRho away from its initial binding 3.4 (averages from two experiments) for unhybridized intact site is that the distal RNA fragment should become prefer- RNA, RNADNA hybrid, and precleaved RNA, respectively entially bound. Moreover, competition experiments and re- (Fig. 3B). Therefore, the 13:1 ratio in Fig. 3A is not due to partitioning controls suggest that this result cannot be ac- rapid intramolecular redistribution, since this could account counted forby either inter- or intramolecular redistribution of Rho following helicase activity. Filter binding provides an indirect measure of Rho-RNA B Ad \osS) | e complexes existing in solution and may be biased by differ- ences in filter retention efficiencies for different RNA spe- ot Tot bd Tot bd 'ot bd Tot ba cies. We therefore based our interpretations not on the 1 2 3 1 2 3 4 5 6 absolute ratios of the T' and HT RNA species bound to Rho, but on differences in this ratio when helicase activity by Rho TV- * HT~ ~~I bound initially to T' is required for generation of the two NO fragments versus control experiments measuring the overall distribution of Rho. If differential retention efficiency of the T' versus HT segments skews the ratio toward the 13-fold FIG. 3. Experiments with excess RNA substrate. A and B are preference for T' seen in Fig. 3A, this must also be true ofthe from the same autoradiograph of a single gel. (A) RNA-RNA duplex substrate. The procedure was the same as in Fig. 2B, except that the RNA-RNA duplex was at 5 nM and Rho was at 1 nM. For clarity, 5' p only the portion ofthe film corresponding to the T' and HT fragments is shown, since the majority of the substrate remained uncleaved. Lane 1, total reaction after incubation for 3 min at 3TC with Rho storage buffer only; lane 2, total reaction after 3 min of incubation with 1 nM Rho; lane 3, RNA retained on the filter by Rho and then eluted. (B) Control experiments. Odd-numbered lanes are from the ATP ADP P total reaction after incubation for a total of 3 min with Rho at 370C; even-numbered lanes display RNA retained on filters by Rho. Lanes 1 and 2, intact unhybridized T'HT RNA at 5 nM incubated with RNase H and 1 nM Rho for 2 min before the addition of oligodeox- /o ynucleotide to 1 juM and incubation for 1 min more; lanes 3 and 4, 5 nM T'HT RNA preannealed to 1 piM oligodeoxynucleotide and p> incubated with 1 nM Rho for 2 min before the addition of RNase H for 1 min; lanes 5 and 6, 5 nM T'HT RNA precleaved with 1 pM oligodeoxynucleotide and RNase H before incubation with.1 nM Rho FIG. 4. A tethered tracking model for Rho helicase activity. The for 3 min. The amounts of the filter-bound and total RNA samples Rho hexamer is represented by a circle, RNA by light lines, and DNA loaded on the gel in both A and B were adjusted to give bands of by double-ended arrows. The Rho binding site on the RNA (trp t') is similar intensity and represent different proportions of the total indicated by a heavy line, and hatched boxes represent RNADNA sample, since Rho is substoichiometric. duplex. Downloaded by guest on September 24, 2021 Biochemistry: Steinmetz and Platt Proc. Natl. Acad. Sci. USA 91 (1994) 1405 experiments in Fig. 3B, where this effect results in at most a (Fig. 4). For further comparison of these models and discus- 3- to 4-fold preference for T'. sion of RNA site connectivity, see ref. 24. Tethered tracking resolves a number ofotherwise puzzling In sum, our results appear incompatible with simple uni- observations. The limiting 1:1 stoichiometry of RNA release directional locomotion of Rho from trp t' to the 3' end of the per Rho hexamer is due to the stable primary binding of Rho RNA and support a tethered tracking mechanism. Further to trp t' RNA. Secondary tracking interactions with the RNA testing of its predictions will require procedures that can extend toward the 3' end of the RNA but are transient and better define the distribution ofRho along RNA molecules in renewable, whereas the primary tethering interaction would solution, elucidate the connectivity pathway ofRNA through be preserved even when the RNA 3' end is reached. This can the Rho hexamer, and examine the interconversion of both explain why ATP hydrolysis continues linearly after helix RNA and ATP binding sites. These in turn should lead to a disruption is complete (7) and why ATPase activation is not deeper understanding of the mechanisms underlying the proportional to the length of RNA extending downstream helicase activity of Rho factor. from trp t' (8). The tethering model also conceptually couples the required sequence-specific interactions with trp t' di- We thank the members ofour laboratory for insights and criticisms rectly to helicase activity, even across intervening RNA throughout the course of this work. We also thank P. H. von Hippel and J. Geiselmann for helpful comments on the manuscript. This sequences of up to 450 nucleotides, which on their own are work was supported in part by National Institutes of Health Grant poor activators of ATP hydrolysis and poor substrates for GM35658 to T.P. and National Institutes ofHealth Genetics Training Rho helicase activity (8). Models that do not incorporate a Grant GM07102 to E.J.S. tethering mechanism must account otherwise for the activa- tion of Rho by initial binding to trp t' to traverse a tract of 1. Platt, T. (1986) Annu. Rev. Biochem. 55, 339-372. inert RNA at least 6 times the length of Rho's 70- to 2. Richardson, J. P. (1990) Biochim. Biophys. Acta 1048,127-138. 80-nucleotide RNA binding site. 3. Platt, T. & Richardson, J. P. (1992) in Transcriptional Regu- Models for Rho action must also incorporate the two-site lation (Cold Spring Harbor Lab. Press, Plainview, NY), pp. model for RNA binding and ATPase activation proposed 365-388. initially by Richardson (10, 11) and recently elaborated upon 4. Chen, C.-Y., Galluppi, G. R. & Richardson, J. P. (1986) Cell 46, 1023-1028. in a "functional dimer" model by von Hippel and coworkers 5. Galloway, J. L. & Platt, T. (1988) J. Biol. Chem. 263, 1761- (17), for which there is cumulative experimental support (3). 1767. With three tight and three weak binding sites for both RNA 6. Brennan, C. A., Dombroski, A. J. & Platt, T. (1987) Cell 48, and ATP, one each on the three asymmetric dimers in the Rho 945-952. hexamer (18-22), the two classes of RNA binding behavior 7. Brennan, C. A., Steinmetz, E. J., Spear, P. & Platt, T. (1990) may be best understood to reflect differential activity of a J. Biol. Chem. 265, 5440-5447. single RNA site per Rho monomer. The three strong RNA 8. Steinmetz, E. J., Brennan, C. A. & Platt, T. (1990) J. Biol. sites are thought to face toward one side of the hexamer, and Chem. 265, 18408-18413. the three weak sites toward the 9. Brennan, C. A. & Platt, T. (1991) J. Biol. Chem. 266, 17296- opposite side, with opposite 17305. polarity (19, 22). 10. Richardson, J. P. (1982) J. Biol. Chem. 257, 5760-5766. A crucial question relates to the connectivity path of an 11. Galluppi, G. R. & Richardson, J. P. (1980) J. Mol. Biol. 138, RNA strand through these sites. One possibility, developed 513-539. in considerable theoretical detail by Geiselmann et al. (23), is 12. Faus, I. & Richardson, J. P. (1990) J. Mol. Biol. 212, 53-66. that the RNA passes directly from the tight site of one dimer 13. Mott, J. E., Grant, R. A., Ho, Y.-S. & Platt, T. (1985) Proc. to the weak site of the next dimer (binding with opposite Natl. Acad. Sci. USA 82, 88-92. polarity), resulting in a pathway through the hexamer alter- 14. Nehrke, K. W., Seifried, S. & Platt, T. (1992) Nucleic Acids nating between strong and weak sites (SWSWSW). In this Res. 20, 6107. 15. Faus, I. & Richardson, J. P. (1989) Biochemistry 28, 3510- model, interconversion between tight and weak RNA- 3517. binding conformations, coupled to the ATP hydrolysis cycle, 16. Schneider, D., Gold, L. & Platt, T. (1993) FASEB J. 7, 201-207. is adopted to generate directed 5'-to-3' motion of Rho along 17. Seifried, S. E., Easton, J. B. & von Hippel, P. H. (1992) Proc. the RNA. Natl. Acad. Sci. USA 89, 10454-10458. The tethered tracking model, while not addressing Rho's 18. Geiselmann, J., Yager, T. D., Gill, S. C., Calmettes, P., Tar- oligomeric structure directly, is more consistent with an dieu, A. & von Hippel, P. H. (1992) Biochemistry 31, 111-120. alternative connectivity scheme, SSSWWW, with significant 19. Geiselmann, J., Seifried, S. E., Yager, T. D., Liang, C. & von implications. It requires that strong-site interactions persist Hippel, P. H. (1992) Biochemistry 31, 121-132. from initial binding through the final release event, with the 20. Stitt, B. L. (1988) J. Biol. Chem. 263, 11130-11137. 21. Geiselmann, J. & von Hippel, P. H. (1992) Protein Sci. 1, weak sites responsible for ATP-dependent tracking along the 850-860. transcript. This predicts that the strong and weak RNA 22. Geiselmann, J., Yager, T. D. & von Hippel, P. H. (1992) binding sites do not interconvert as ATP is hydrolyzed and Protein Sci. 1, 861-873. that an RNA segment of increasing size should become 23. Geiselmann, J., Wang, Y., Seifried, S. E. & von Hippel, P. H. transiently looped out between the strong and weak binding (1993) Proc. Natl. Acad. Sci. USA 90, 7754-7758. faces as the weak interactions extend farther along the RNA 24. Platt, T. (1994) Mol. Microbiol., in press. Downloaded by guest on September 24, 2021