Functional coupling between a distal interaction and the cleavage site in bacterial...

doi:10.1016/j.jmb.2011.05.049 J. Mol. Biol. (2011) 411, 384–396

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

Functional Coupling between a Distal Interactionand the Cleavage Site in BacterialRNase-P-RNA-Mediated Cleavage

Shiying Wu1, Yu Chen2, Magnus Lindell1, Guanzhong Mao1

and Leif A. Kirsebom1⁎1Department of Cell and Molecular Biology, Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden2Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA

Received 18 March 2011;received in revised form31 May 2011;accepted 31 May 2011Available online12 June 2011

Edited by J. Doudna

Keywords:RNase P;ribozyme;divalent metal ions;tRNA precursors;tRNA processing

*Corresponding author. E-mail [email protected] used: RPR, RNase

loop; TBS, TSL binding site; S-domaC-domain, catalytic domain; pre-tRNwt, wild type; Eco, Escherichia coli.

0022-2836/$ - see front matter © 2011 E

Bacterial RNase P consists of one protein and one RNA [RNase P RNA(RPR)]. RPR can process tRNA precursors correctly in the absence of theprotein. Here we have used model hairpin loop substrates corresponding tothe acceptor, T-stem, and T-loop of a precursor tRNA to study theimportance of the T-loop structure in RPR-alone reaction. T-stem/loop(TSL) interacts with a region in RPR [TSL binding site (TBS)], forming TSL/TBS interaction. Altering the T-loop structure affects both cleavage siteselection and rate of cleavage at the correct site +1 and at the alternative site−1. The magnitude of variation depended on the structures of the T-loopand the TBS region, with as much as a 150-fold reduction in the rate ofcleavage at +1. Interestingly, for one T-loop structure mutant, no differencein the rate at −1 was detected compared to cleavage of the substrate with anunchanged T-loop, indicating that, in this case, the altered T-loop structureprimarily influences events required for efficient cleavage at the correct site+1. We also provide data supporting a functional link between a productiveTSL/TBS interaction and events at the cleavage site. Collectively, ourfindings emphasize the interplay between separate regions upon formationof a productive RPR substrate that leads to efficient and accurate cleavage.These new data provide support for an induced-fit mechanism in bacterialRPR-mediated cleavage at the correct site +1.

© 2011 Elsevier Ltd. All rights reserved.

Introduction

tRNA genes are transcribed as precursors that areprocessed at both the 5′ end and the 3′ end tobecome functional tRNAs. The ribonucleoproteinRNase P is responsible for trimming the 5′ termini toproduce tRNAs with phosphate at their 5′ ends.

ress:

P RNA; TSL, T-stem/in, specificity domain;A, precursor tRNA;

lsevier Ltd. All rights reserve

RNase P consists of one RNA [RNase P RNA (RPR)]and one or more proteins, depending on the origin: 1protein in bacteria, at least 4 or 5 proteins in archaea,and 9 or 10 proteins in eukarya.1–5 Regardless oforigin, RPR alone, without protein, is capable ofcleaving various substrates.6–10 Recent reports,however, demonstrate protein-based RNase-P-likeactivity.11,12

On the basis of its secondary structure, RPR isdivided into two major domains: specificity domain(S-domain) and catalytic domain (C-domain).13

Available crystal structures suggest that the S-domainis folded similarly with or without the C-domain.14–18

The S-domain contains the binding site for the T-stem/loop (TSL) region of precursor tRNA (pre-tRNA).This site is referred to as the TSL binding site (TBS),

d.

mailto:[email protected]

http://dx.doi.org/10.1016/j.jmb.2011.05.049

385RNase P Cleavage and tRNA Precursor Processing

and the interaction between the two is called TSL/TBS interaction.1–4

Model hairpin substrates have been useful inidentifying factors that influence substrate interac-tion, cleavage efficiency, and selection of thecleavage site.1,2 We recently reported that replacingthe T-loop in the model hairpin loop substratepATSerCG (Fig. 1) with a GAAA tetraloop resultedin significant cleavage at an alternative position, −1(i.e., between residue −1 and residue −2). Thismiscleavage at −1 was suppressed by altering thestructure of the TBS region such that cleavageoccurred preferentially at the correct site +1 (Fig. 1;see also the text below).10,22 On the basis of thesedata, we provided a model in which a productiveTSL/TBS interaction influences the positioning ofchemical groups and/or Mg2+ at the cleavage site.22

Fig. 1. Structure of the different hairpin loop modelsubstrates used in the present study. The originalpATSerCG substrate is derived from an E. coli tRNASer

precursor, and the numbering in the vicinity of thecleavage site refers to the numbering of tRNAprecursors.19,20 The canonical cleavage site betweenresidue −1 and residue +1 is marked with an arrow. Thedifferent tetraloop substrates, where the “T-loop” (gray)had been replaced by GAAA, UUCG, and CUUG,respectively, carried a U-to-C substitution at −1, asindicated. A C at −1 results in likely pairing with G+73(corresponding to the tRNA discriminator base).21 Theencircled residues in the T-loop correspond to residuesC56 and A57 in tRNA (for further details, see Discussionand Fig. 6). The 2′OH was substituted with 2′H at G+1 inpATSerCGGAAA (for details, see the main text).

Given that the GAAA tetraloop adopts a character-istic structural topography,23–26 we decided tochange its structure and to study the effect oncleavage. Moreover, replacement of the T-loop witha GAAA tetraloop results in an increased require-ment for Mg2+ and a reduction in the rate ofcleavage at a suboptimal Mg2+ concentration.22

Therefore, a second objective was to investigate towhat extent the absence of a productive (or altered)TSL/TBS interaction affects the rate of cleavage inthe presence of an optimal concentration of Mg2+.Finally, we inquired whether structural changes inRPR in the vicinity of the cleavage site couldcompensate for a nonproductive TSL/TBS interac-tion. If so, this would be evidence for a functionallink between the TSL/TBS interaction and events atthe cleavage site. Here we report that altering thestructure of the TSL/TBS interaction influencesevents at the cleavage site to varying extents,depending on the structure of the tetraloop in thesubstrate. Importantly, our new data show thatreplacement of the T-loop with a GAAA tetraloopaffects the rate of cleavage at the correct site +1,while no change in the rate at the alternative site −1was detected. Our data also provide evidence for afunctional link between a productive TSL/TBSinteraction and the binding of the 3′ end of thesubstrate, as well as the 2′OH of G+1 in the substrate.These new data will be discussed in terms of aninduced-fit mechanism in bacterial RPR-mediatedcleavage at the correct site +1.

Results

Miscleavage of model hairpin loop substrateswith alternative loop structures

The GAAA tetraloop adopts a specific structuralfold that is distinct from the structures of othertetraloops such as the UUCG and CUUG tetraloops(Fig. 1).23–26 These structural differences alter thepositioning of hydrogen-bond donors and accep-tors, possibly affecting interaction with RPR and,thereby, cleavage. Therefore, we generatedpATSerCGUUCG and pATSerCGCUUG (Fig. 1).These two substrates, pATSerCG (which has anintact T-loop) and pATSerCGGAAA, were subjectedto cleavage by wild-type (wt) Escherichia coli (Eco)RPR. First, we analyzed if these two new substrateswere also cleaved at the alternative site −1. Cleavagesite selection was inferred from the mobilities ofthe 5′ cleavage fragments, and the data are shownin Fig. 2.Consistent with our previous data,10 ,22

pATSerCGGAAA was preferentially cleaved at −1by wt EcoRPR, while pATSerCG was cleaved with amuch lower frequency at this site under theseconditions. Replacing the GAAA tetraloop with

Fig. 2. Cleavage of the differenttetraloop substrates by various RPRderivatives. (a) The reactions wereperformed in buffer C containing800mMMg2+ at 37 °Cwith differentRPRs, as indicated. Lane 1,p A T S e r C G U U C G ; l a n e 2 ,p A T S e r C G C U U G ; l a n e 3 ,pATSerCGGAAA; lane 4, pATSerCG.The positions of the 5′ cleavagefragments generated after cleavagewith the different RPRs are markedwith +1 and −1, respectively. Con-trols refer to substrates incubatedwithout RPR. The concentrations ofthe RPR variants and substrateswere 0.4μMand0.02μM, respectively.(b) Cleavage of pATSerCGGAAA carry-ing a 2 ′H at G+ 1. Lane 1,pATSerCGGAAA (all-ribo); lane 2,pATSerCGGAAA with a 2′H at G+1.The percent cleavage at −1 is theaverage of at least two independentexperiments,witherrorsas follows: wt(pATSerCGGAAA), 76±0.03; wt(pATSerCGGAAA with 2′H at G+1),54±1.3; RPRC294 (pATSerCGGAAA),37±0.5; RPRC294 (pATSerCGGAAAwith 2′H at G+1), 17±0.02; RPRG235(pATSerCGGAAA), 6±0.08; RPRG235(pATSerCGGAAA with 2′H at G+1),2.6±0.3.

386 RNase P Cleavage and tRNA Precursor Processing

CUUG and UUCG also resulted in cleavage at −1.However, the frequency of cleavage at the alterna-tive site −1 was reduced compared to that ofpATSerCGGAAA. The substrate with the lowestfrequency of cleavage at −1 was the UUCG variant(Figs. 2 and 3). Moreover, under single-turnoverconditions, wt RPR cleaved the CUUG and UUCGtetraloop substrates at much lower rates comparedto the cleavage of pATSerCG and pATSerCGGAAA(Table 1; see also the text below).The reaction catalyzed by RPR requires the

presence of divalent metal ions, preferentially Mg2+,and, depending on the substrate, the concentrationrequired for optimal cleavage varies.22 We therefore

analyzed the cleavage of the different pATSerCGvariants as a function of Mg2+ concentration. Asshown in Fig. 4, optimal cleavage was reached at amuch higher Mg2+ concentration for the tetraloopsubstrates compared to pATSerCG. Moreover, thecleavage site distribution for neither of the tetraloopsubstrate variants did not vary to any significantextent with the Mg2+ concentration. The one excep-tion was pATSerCGUUCG, which was miscleaved witha slightly higher frequency at −1 at 75 mM and150 mM Mg2+ (47% and 40%, respectively, versus≈30% between 300 mM and 1200 mM). ForpATSerCG, we observed an increase in cleavageat −1 at a higher Mg2+ concentration, but not to the

image of Fig. 2

Fig. 3. Frequency of miscleavageat −1 for the various RPRs cleavingthe different substrates, as indicated.The experiment was performed at37 °C in buffer C containing 800 mMMg2+. The final concentration ofRPRranged between 0.4 μM and 4 μM,while the substrate concentrationwas ≈0.02 μM. The incubation timefor the reactions varied dependingon RPR–substrate combination.


level observed for the tetraloop substrates (Figs. 3and 4). This is in contrast to the cleavage performedat a low Mg2+ concentration (40 mM), wherecleavage at −1 was 2.4%.19 In summary, thesedata suggest that the loop structure of hairpinmodel substrates affects the selection of the cleavagesite and the Mg2+ requirement.

Cleavage as a function of changes in RPR

The crystal structure of RNase P in complex withtRNA reveals that the TSL region of tRNA interactswith residues in the S-domain.18 Specifically, resi-dues corresponding to A129, A130, A180, G229, G230,U231, A232, and A233 (highlighted in red in Fig. 5a) inEcoRPR interact with the TSL region. Substitution ofresidues G125 and/or C235 (base pair marked ingreen; Fig. 5) alters the structure in the vicinity ofTBS.22 This structural change suppresses the cleav-age of pATSerCGGAAA at −1 such that the cleavageoccurs preferentially at +1 (see, e.g., Figs. 2 and 3).We have argued that this is due to an altered TSL/TBS interaction (or lack of a productive TSL/TBS

Table 1. Apparent rates (kapp; picomoles cleaved per minuEcoRPRG235, and EcoRPRC294

Substrate Site wt

pATSerCG +1 2.5±0.16−1 0.41±0.033

pATSerCGGAAA +1 0.15±0.04−1 0.50±0.14

pATSerCGCUUG +1 0.021±0.0057−1 0.01±0.002

pATSerCGUUCG +1 0.025±0.0055−1 0.01±0.0017

ND=not determined.The experiments were performed in buffer C containing 800mMMg2+

0.02 μM, respectively. Given values are averages of at least two indep

interaction).22 We therefore inquired whether RPRswith structurally altered TBS regions (Fig. 5) alsocleave pATSerCGUUCG and pATSerCGCUUG prefer-entially at +1. Consistent with our previous data, theRPR variants (EcoRPRC125, EcoRPRG235, andEcoRPRC125/G235) cleaved pATSerCGGAAA mainlyat the correct site +1 (Figs. 2 and 3). From these data,it is also apparent that the three variants cleavedpATSerCGUUCG and pATSerCGCUUG with a fre-quency of more than 90% at +1. We also tested anRPR variant where A124 was replacedwith U. Again,an increase in cleavage at +1 was detected, althoughto a lesser degree compared to changing the base pair125/235 (Fig. 3). The Mg2+ profiles for the differenttetraloop substrates using wt RPR and RPRG235 weresimilar (Fig. 4). Moreover, RPRG235 cleaved theUUCG and CUUG tetraloop substrates at reducedrates at 800 mMMg2+ (Table 1). However, relative towt RPR, RPRG235 cleaved all three tetraloop sub-strates at +1 with faster rates. These data emphasizethat the structural topography (Fig. 5b) at and in thevicinity of TBS not only plays a role in site selectionbut also has an effect on the rate of cleavage at +1.

te) for the cleavage of pATSer variants with EcoRPRwt,

EcoRPR variant

G235 C294

0.74±0.062 4.3±0.11ND 0.52±0.02

0.55±0.052 0.98±0.0490.016±0.0022 0.58±0.0030.052±0.012 0.16±0.00520.0018±0.001 0.02±0.000350.054±0.004 0.14±0.046

ND 0.036±0.014

at 37 °C. The concentrations of RPRs and substrates were 4 μMandendent experiments.

image of Fig. 3

Fig. 4. Cleavage of the various model substrates by EcoRPR as a function of Mg2+ under single-turnover conditions atpH 6.1 (buffer C) and 37 °C. The data reported represent the average of at least two independent experiments.


The tetraloop structure affects theHill coefficient

With the Mg2+ titration experiments, we were ableto calculate the Hill coefficients for the cleavage ofthe different pATSerCG variants for both wt RPRand RPRG235 (Table 2). Except for the cleavage of theGAAA tetraloop substrate with wt EcoRPR, calcu-lation of the Hill coefficients suggested a minimalrequirement of three Mg2+ binding sites to stabilizethe wt RPR–substrate complex. In contrast, the Hillcoefficient for the cleavage of pATSerCGGAAA at +1increased from roughly 3 to ≈4, indicating that thestructure of the loop influences the stabilization ofthe wt RPR complexes by affecting the occupation ofcritical Mg2+ binding sites. An increase from 3 to 4was also detected for all substrates, except forpATSerCGGAAA, when the wt was replaced withthe G235 variant. This suggests that the alteredstructure in the TBS region changes the number ofMg2+ binding sites needed to stabilize the RPR–substrate complex, and that this depends on thestructure of the tetraloop. In view of cleavage at −1,

the Hill coefficient increased from ≈2 to almost 5 forpATSerCGGAAA compared to pATSerCG, while theincrease was not as pronounced for the othertetraloop substrates. Hence, this suggests that thenumber of critical Mg2+ binding sites for cleavage at+1 and the number of critical Mg2+ binding sites forcleavage at −1 differ, and that this difference isrelated to the structure of the loop. In this context, ithas been suggested that the S-domain of Pyrococcusfuriosus RPR interacts differently with the T-loop ofmodel hairpin loop substrates compared to EcoRPR.It is therefore noteworthy to mention that the Hillcoefficient in the cleavage of pATSerUG by P.furiosus RPR was recently determined to be ≈4.10

Interaction between the 3′ end of the substrateand RPR suppresses cleavage at −1

Residue G+73 in the substrate interacts with U294in the RPR–substrate complex (referred to as the+73/294 interaction;29 see also Reiter et al.18).Replacing U at position 294 with C seems tofacilitate the disruption of the C−1/G+73 pair.19,30

image of Fig. 4

Fig. 5. Illustrations of the predicted secondary structures of EcoRPR (M1 RNA)27,28 and a three-dimensional structureview of the RPR region that interacts with the TSL region of the substrate.18 (a) The S-domain and the C-domain areseparated by a demarcation line. Residues in the S-domain (P11) that are in close contact with the TSL region of thesubstrate are highlighted in red. Substituted residues are marked in green, and the corresponding RPRs are referred to asRPRU124, RPRC125, RPRG235, RPRC125/G235, and RPRC294 (for details, see the text). The red arrows indicate GNRAreceptors. (b) Part of the TSL/TBS interaction as revealed by the crystal structure of RPR in complex with tRNA.18 Thebase pair G125/C235 is highlighted in green; G19 of tRNA and A130 of RPR are marked with arrows (for details, see Fig. 6).


We argued that the RPRC294 variant would thereforecleave the tetraloop substrates with a higherfrequency at +1 relative to the wt. To test this, wecleaved the different substrates with RPRC294. Asshown in Fig. 3, it is apparent that this change inRPR increased the frequency of cleavage at +1 andeven more so for the tetraloop variants. Thisincreased frequency is associated with an increasein the rate of cleavage at +1, while only minorchanges in the −1 cleavage rates were detected(Table 1; see also the text below). With respect to siteselection, changing the structure of the +73/294interaction can thus compensate, to some extent, fora less productive TSL/TBS interaction. As observed

for the other RPR variants (see the text above),RPRC294 required a higher Mg2+ concentration toreach the optimal efficiency of the cleavage ofpATSerCGGAAA compared to pATSerCG. More-over, RPRC294 cleaved pATSerCG more frequentlyat −1 with increasing Mg2+ concentrations, as wasobserved for wt RPR (see the text above). However,the level of miscleavage at −1 was lower comparedto the case of wt RPR. The miscleavage ofpATSerCGGAAA at −1 did not change to anysignificant degree with the Mg2+ concentration, aswas observed for the other RPR variants (data notshown). Interestingly, the Hill coefficient forRPRC294 in the cleavage of either pATSerCG or

image of Fig. 5

Scheme 1. Simplified reaction scheme for cleavage at+1 and at −1.


pATSerCGGAAA was estimated to be approximately3 (except for the cleavage of pATSerCG at −1, whichis one unit higher or at least two units higher,respectively, than the cleavage of pATSerCG withwt RPR at −1) (Table 2). Together, these data suggestthat strengthening the +73/294 interaction compen-sated for a less productive (or nonproductive) TSL/TBS interaction. In addition, substitution of U294with C in RPR affects the number of critical Mg2+

binding sites for cleavage at −1. Conceivably, this isdue to the fact that the U-to-C change affects Mg2+

binding near the +73/294 interaction (see also thetext below).2

Given that residue G+1 in the substrate is close tothe scissile bond, we also tested whether thesubstitution of its 2′OHwith 2′H affects the selectionof the cleavage site. Hence, pATSerCGGAAA carry-ing a 2′H at position +1 (Fig. 1) was subjected tocleavage with wt RPR, RPRC294, and RPRG235. Asshown in Fig. 2b, the 2′H at +1 results in a lowerfrequency of cleavage at −1 compared to the casewith the all-ribo pATSerCGGAAA, irrespective ofRPR. This finding provides additional evidence for afunctional coupling between events at the cleavagesite and the TSL/TBS interaction.

Influence of TSL/TBS interaction on the rateof cleavage

Cleavage of model substrates can be describedaccording to the simplified Scheme 1, where kobs andkobs* reflect the rates of cleavage at +1 and −1,respectively, as indicated. We argue that, underthese conditions (800 mM Mg2+), Ksto≈Kd, sincek−1≫kobs in the cleavage of pATSerUG by EcoRPR,where k−1 corresponds to the rate of dissociation(Fig. S1).10,31,32 Moreover, ES1 to ES1⁎ describes, forexample, the positioning of Mg2+ that results incleavage at −1, while ES1 to ES2 accounts for, forexample, the opening of C−1/G73 (when present)

Table 2. Hill coefficients for the cleavage of pATServariants with different RPRs, as indicated

Substrate Site

EcoRPR variant

wt G235 C294

pATSerCG +1 3.4±0.46 3.7±0.17 3±1.4−1 1.7±0.73 ND 4.5±0.78

pATSerCGGAAA +1 3.7±0.37 3.8±0.21 3.5±0.32−1 4.6±0.24 ND 3.2±0.22

pATSerCGCUUG +1 3.1±0.05 4.1±0.19 ND−1 3.2±0.22 ND ND

pATSerCGUUCG +1 2.7±0.04 4.1±0.1 ND−1 2.4±0.23 ND ND

ND=not determined.The Hill coefficients were calculated as outlined in Materials andMethods. Each value is the average of several independentexperiments.

and the positioning of Mg2+ resulting in cleavage at +1.Note also that there is no precedent for the reversereaction [i.e., formation of ES2 from EP (or from EP⁎ toES1⁎)], hence k−3 (and k−3⁎) and the arrows are placed inparentheses. On the basis of the Mg2+ profiles (Fig. 4),the previously reported kinetic constants for thecleavage of pATSerCG and pATSerCGGAAA with wtRPR and RPRG235 were determined at the suboptimalMg2+ concentration of 160 mM.22 Hence, here wedetermined kobs and kobs/K

sto for the cleavage of thedifferent tetraloop substrates and pATSerCG undersingle-turnover conditions at pH6.1 and 800mMMg2+.The data are presented in Table 3.

Structure of the tetraloop and its effect on thekinetic constants kobs and kobs/K

sto

For cleavage of pATSerCG at the correct site +1,kobs/K

sto is approximately 5-fold lower compared topATSerUG (Table 3; data for pATSerUG were takenfrom Sinapah et al.10). The reduced cleavageefficiency is due to an ≈3-fold decrease in kobs,while binding (Kd) is affected by a factor of 2. Thesedata are in agreement with our reported kineticanalysis at 160 mM Mg2+ using these twosubstrates.22 As we have discussed elsewhere, thedrop in the cleavage efficiency for pATSerCG ispartly due to the presence of the C−1/G73 pair,which has to be broken before cleavage at +1 (S.W.et al., unpublished).19 Interestingly, our presentexperiments at 800 mM Mg2+ also allowed us tocompare the kinetic constants for the cleavage ofpATSerCG at +1 and at the alternative site −1. Thiscomparison could not be reliably performed in theprevious study.22 The new experiments revealed an≈5-fold difference in kobs/K

sto for cleavage at +1versus −1 that is attributed to a reduction in kobs.Given that Ksto (Kd) did not change, this suggeststhat the binding affinity is the same and does notdepend on which cleavage site is selected.Replacing the T-loop with the GAAA tetraloop

resulted in a 15-fold reduction in kobs at +1, while Kdchanged less than 2-fold compared to pATSerCG,irrespective of whether the −1 or the +1 cleavage sitewas chosen. This should be compared to the 100-

image of Scheme 1

Table 3. The kinetic constants kobs and kobs/Ksto for the cleavage of different pATSerCG and tetraloop variants with RPRs,

as indicated

RPR/substrate Site kobs (min−1) kobs/Ksto (min−1 μM−1) Kd (μM)

wt EcoRPR/pATSerUG +1 12±1.3a 19±3.8a 0.63−1

wt EcoRPR/pATSerCG +1 4.9±1.0 3.9±2.1 1.3−1 0.98±0.079 0.71±0.28 1.4

wt EcoRPR/pATSerCGGAAA +1 0.33±0.042 0.16±0.023 2.0−1 1.1±0.05 0.45±0.14 2.4

wt EcoRPR/pATSerCGUUCG +1 0.11±0.01 0.03±0.003 3.7−1 0.038±0.0042 0.027±0.0063 1.4

wt EcoRPR/pATSerCGCUUG +1 0.03±0.001 0.005±0.0001 6.0−1 0.02±0.003 0.0032±0.00026 6.3

EcoRPRG235/pATSerCG +1 1.9±0.14 0.47±0.015 4.0−1 ND ND ND

EcoRPRG235/pATSerCGGAAA +1 2.0±0.12 0.42±0.38 4.8−1 ND ND ND

EcoRPRC294/pATSerCG +1 7.2±0.96 4.2±0.85 1.7−1 0.71±0.07 0.73±0.13 0.97

EcoRPRC294/pATSerCGGAAA +1 2.3±0.08 0.86±0.012 2.7−1 1.6±0.11 0.56±0.014 2.9

ND=not determined.The experiments were performed under saturating single-turnover conditions at 800 mMMg2+ and pH 6.1, as described in Materials andMethods. The final concentration of the substrate was 0.02 μM. The concentrations of the different RPR variants were varied between0.1 μM and 51.2 μM, and the concentration range varied depending on RPR and substrate.

a Data taken from Sinapah et al.10


fold decrease in kobs at 160 mM, as we reportedpreviously (cf. Table 2 in Brännvall et al.22). Thisdifference emphasizes the importance of Mg2+

concentration when the cleavage rates of differentsubstrates are compared. Interestingly, no change inkobs for cleavage at −1 was detected compared tocleavage of pATSerCG at this position. Thus, thepresence of the GAAA tetraloop appears to primar-ily affect events required for efficient cleavage at thecorrect site +1.For the substrates with UUCG or CUUG tetra-

loops, both kobs and kobs/Ksto were reduced com-

pared to pATSerCGGAAA, where pATSerCGCUUG iscleaved with the lowest efficiency. This is true forcleavage at −1, as well as for cleavage at +1. In thecase of cleavage at +1, kobs was reduced 10-fold (cf.cleavage at +1; pATSerCGGAAA versuspATSerCGCUUG;Table 3), while in comparison to cleavage ofpATSerCG, it was N150-fold lower. From the Kdvalues, it is also apparent that pATSerCGCUUG bindswith lower affinity to EcoRPR than to the othersubstrates (cf. ≈6-fold difference for pATSerCGversus pATSerCGCUUG). Moreover, a comparison ofthe Kd values for the cleavage of pATSerCGUUCG at−1 and the cleavage of pATSerCGUUCG at +1revealed a 2.6-fold difference. These data suggestthat the tetraloop structural topography (and therebythe TSL/TBS interaction) influences the rate andefficiency of cleavage at the correct site and at thealternative site −1. We therefore conclude that theloop structure of the model substrate affects theinteraction between the RPR and the substrate (i.e.,the TSL/TBS interaction). Disrupting or altering this

interaction subsequently results in changes in the rateof cleavage at the correct cleavage site +1. However,the effect on the rates (kobs) at +1 and −1 dependson the structure of the tetraloop and, ultimately, onthe way it interacts with RPR.

Altering the structure of the TBS region and itseffect on kobs and kobs/K

sto

Determination of the kinetic constants for theRPRG235 variant at 800 mM Mg2+ revealed that kobsand kobs/K

sto were the same for both of thesesubstrates (cf. pATSerCG and pATSerCGGAAA;Table 3). Compared to the cleavage of pATSerCGby wt RPR at +1, the kobs for RPRG235 was only2-fold lower. At the same time, kobs/K

sto was reducedby a factor of ≈10, resulting in an almost 4-folddifference in Kd. This should be compared to thealmost 10-fold and 40-fold differences in kobs andkobs/K

sto, respectively, at 160 mM Mg2+ (cf. Table 2in Brännvall et al.22). Hence, the data reported hereare in keeping with our previous findings, but thedifference from wt RPR is less pronounced at800 mM Mg2+. Given that Ksto≈Kd, we can nowalso conclude that the structural change in the TBSregion has a modest effect on both binding and rateof cleavage (kobs).A comparison of the kinetic constants for the

cleavage of pATSerCG and pATSerCGGAAA withRPRG235 shows that kobs and kobs/K

sto did notchange (Table 3). This is in contrast to the casewith wt RPR, as discussed above. It is alsonoteworthy that RPRG235 cleaved pATSerCGGAAA


more efficiently than wt RPR at the correct site+1: kobs increased almost 10-fold, while thechanges in kobs/K

sto and Kd were 2-fold to 3-fold,respectively. Thus, with respect to the cleavage of theGAAA tetraloop substrate, the C-to-G change atposition 235 should be considered as a suppressormutation.Together with the data discussed above, these

findings agree that substituting the C at position 235with G distorts the TBS structure, and that thisstructural change affects the TSL/TBS interactionand events at the site of cleavage.

Alteration of the interaction between RPR andthe 3′ end of the substrate: Effects on kobsand kobs/K

sto

Given that the RPRC294 variant cleaved the tetra-loop substrates with increased rates at +1 comparedto wt RPR, we inquired whether this could be due toa change in kobs and/or kobs/K

sto (or Kd). Hence, wedetermined kobs and kobs/K

sto for pATSerCG andpATSerCGGAAA and used these values to calculateKd, as discussed above (see Table 3).For cleavage of pATSerCG, kobs increased less

than 2-fold, while kobs/Ksto did not change much for

RPRC294 compared to wt, irrespective of whether wecompared cleavage at +1 or at −1. However, the kobsfor the cleavage of the tetraloop substratepATSerCGGAAA at +1 with RPRC294 was 7-foldhigher compared to the cleavage with wt RPR.Interestingly, the kobs value for RPRC294 was thesame as that determined for RPRG235. With respectto the cleavage of pATSerCGGAAA at −1, kobs wasonly slightly enhanced when comparing RPRC294with wt RPR. We also note that the overall rate (i.e.,considering cleavage at both positions) was ≈2.6-fold higher in the cleavage of pATSerCGGAAA withRPRC294 compared to wt RPR, while it was just≈30% higher in the case of the cleavage ofpATSerCG. Moreover, a comparison of kobs/K

sto

for the RPRC294 and wt RPR cleavage of the GAAAtetraloop substrate at −1 did not reveal anysignificant difference, while cleavage at +1 withRPRC294 gave a N5-fold increase. Hence, calculationof Kd revealed similar (or slightly higher) values forRPRC294 compared to the wt, indicating no signifi-cant change in binding affinity.These data suggest that strengthening the +73/

294 interaction in the vicinity of the cleavage siteenhances the rate of cleavage (kobs). This occursmainly at the correct site +1, irrespective ofsubstrate, but even more so in the case ofpATSerCGGAAA (see also Table 1). Together, thesedata suggest that a stronger interaction between the3′ end of the substrate and RPR can compensate foran altered TSL/TBS interaction and hence provide afunctional link between binding to the TSL regionand events at the cleavage site.

Discussion

The structural topography of the interactionbetween the TSL region of pATSer model substratesand the RPR TBS region affects both cleavage siterecognition and rate of cleavage at the correct site+1 (Brännvall et al.22 and this work). However, ournew data show that the magnitude of the changesin rate and site selection is dependent on thestructure of the loop. Importantly, as reported here,the change in the rate is related to the correct site+1, while the cleavage rate at −1 comparingpATSerCG and pATSerCGGAAA is unchanged.Interestingly, altering the GAAA tetraloop toCUUG or UUCG increased the frequency ofcleavage at +1, and this was accompanied bylower rates of cleavage at both positions. Moreover,these effects can be compensated for either byaltering the structure in the TBS site in RPR or bystrengthening the interaction between the 3′ end ofthe substrate and RPR in the vicinity of the cleavagesite (i.e., the +73/294 interaction; this work).Changing the +73/294 interaction suppresses theeffect induced by a nonproductive TSL/TBS inter-action and provides a functional link between thesetwo interactions in the RPR–substrate complex. Afunctional link between the TSL/TBS interactionand the cleavage site is further emphasized by theobservation reported here that substitution of the2′OH at G+1 in the substrate with 2′H alsosuppressed miscleavage at −1 to a low extent.These findings support an induced-fit mechanismin RPR-mediated cleavage.22 The structural topog-raphy of the +73/294 interaction can influence thecleavage and binding of functionally importantmetal(II) ions (e.g., Mg2+) at and in the vicinity ofthe cleavage site.19,29,30,33–38 This therefore raises thepossibility that a productive TSL/TBS interaction,required for efficient cleavage at the correct site,involves the positioning of metal(II) ions near thecleavage site (see the text below). In this context, wealso note that increasing the size of the T-loop inSELEX (Selective Evolution of Ligands by EXpo-nential enrichment)-evolved substrates lowers thecleavage efficiency (kcat/Km) for EcoRPR.

39

The crystal structure of the complex betweentRNA and RNase P reveals structural details of theTSL/TBS interaction (Fig. 6). Residues G19 and C56in the TSL stack on A112 and G147, respectively,and this results in stabilization of the TSL/TBSinteraction.18 In EcoRPR, A112 and G147 corre-spond to A130 and A180, respectively (Fig. 5).Thermus thermophilus RPR also has A at thesepositions (A120 and A172), where they are posi-tioned more or less in the same way as A112 andG147 (Fig. 6) in the structure of the T. thermophilusS-domain.15 Hence, we expect that, relative to G19and C56 (in both tRNA and their precursors), A130and A180 in EcoRPR are positioned essentially in

Fig. 6. Crystal structures of the TBS region (a), the TSL/TBS interaction (b), and amodel of the RNase P cleavage site(c). (a) The structure of the TBS region in the S-domain in theabsence of substrate.15 Residues A130 and A180 (E. colinumbering) are indicated. The gray balls correspond tometal(II) ions. (b) The structure of the TBS region with tRNA.18

Interacting residues are marked, and residue numbers givenin parentheses refer to E. coli numbering (see Fig. 5a). (c) Thisstructure was taken from Deng et al.40 and corresponds to apart of the signal recognition particle RNA structure (ProteinData Bank ID: 1LNT). Residues C−1, G+1, C+72, and G+73 aremarked. The sphere in gold represents theMg2+ that activatesthe nucleophile (H2O), and the arrowsmark the distance to P(+1) and P(−1) (for details, see the text).


the same way as shown in Fig. 6b. Assuming thatthe T-loop of the model hairpin loop substratesadopts a structure similar to that of the T-loop in atRNA, then the residue corresponding to C56 (seeFig. 1) can still stack on A180. It is possible that theA residue at position “57” (analogous to position 57in tRNA) in the T-loop of the model substrate stackson A130 because G19 is absent. This would give asimilar TSL/TBS interaction for the model hairpinloop substrates and tRNA, and explain why thistype of model substrate interacts productively withthe TBS region. Taken together, stacking is sug-gested to play a major role for a productive TSL/TBS interaction. Replacing the T-loop with atetraloop disrupts or alters this interaction andinfluences cleavage, as reported here andelsewhere.22 In addition, our new findings showthat cleavage efficiency and cleavage site selectiondepend on the structure of the tetraloop. Moreover,as exemplified by EcoRPR, the different GNRAtetraloops dock to two consecutive base pairs. Thecomposition and structure of these 2 bp differ(marked with red arrows in Fig. 5a) depending onthe structure of the tetraloop. Note also that aninternal loop, together with two C-G pairs, acts as areceptor for GNRA tetraloops.41,42 The differenttetraloops may therefore interact differently withthe TBS region. As a consequence, this results inloss of stacking and less productive TSL/TBSinteractions that give differential effects at theactive center. Changing the structure of base pair125/235 affects the interaction between the tetra-loops and TBS. This is not simply due to disruptionof this base pair because changing its orientation(G125/C235 to C125/G235) gave essentially the samephenotype as if it were disrupted.22 This mightindicate that the G at position 235 prevents theformation of a less productive TSL/TBS interactionand promotes an alternative that mimics theinteraction between the T-loop and wt RPR.Alteration of the 125/235 pair might even abolishthe formation of an interaction between TSL andTBS, but the effects seen when the tetraloopstructure was altered argue against this. Eitherway, our data clearly show that the distal TSL/TBSinteraction affects events at the cleavage site. This isfurther accentuated by our finding that strengthen-ing the +73/294 interaction can compensate for aless productive TSL/TBS interaction (see also thetext below). It should be noted that the precursor to4.5S RNA (pre-4.5S RNA) is a hairpin substrate thatis structurally different from pre-tRNAs. Interest-ingly, earlier data suggest that EcoRPR processespre-4.5S RNA with less efficiency relative to pre-tRNA, mainly due to a lower affinity for pre-4.5SRNA.43 This might be the result of a distorted TSL/TBS interaction, since the T-loop is missing and/orthe distance (which normally is 12 bp in pre-tRNAs)between the cleavage site and residues showing


some resemblance to the TSL region in pre-tRNA isincreased from 12 bp to 14 bp.Distal contacts in other systems have also been

shown to influence catalytic rates. For example, acomparison of the structure of the minimal ham-merhead RNA with that of Schistosoma mansonireveals that a peripheral loop–bulge contact en-hances the catalytic rate by at least 50-fold.44–46

Likewise, correct decoding gives a faster rate ofGTPase activation where a conformational changeinduced by a correct codon–anticodon interactionplays an important role.47,48 Thus, for RNA-basedreactions, distal RNA–RNA interactions are com-mon and play an important role in efficiency andcatalysis, as well as in cleavage at the correct site.Although we do not yet have information on how

a productive TSL/TBS interaction structurally af-fects events at and in the vicinity of the cleavage site,our new data give some insights. First, the rate ofcleavage (kobs) did not change for pATSerCG andpATSerCGGAAA at −1. Second, G+73 in the substrateinteracts with U294 (referred to as the 73/294interaction).18,29,49 The RPR variant with C atposition 294 resulted in suppression of cleavage at−1, as well as enhancement of the rate of cleavage at+1 (Fig. 3 and Table 3). This suggests that alteringthe 73/294 interaction compensates, to some extent,for the lack of a productive TSL/TBS interaction.Together, these data are consistent with a modelwhere the TSL/TBS interaction affects the disrup-tion of the C−1/G+73 pair in the substrate, which is aprerequisite for exposure of and cleavage at thecorrect site.19,29,49,50 This does not exclude thepossibility that the TSL/TBS interaction facilitatesthe formation of the +73/294 interaction. Conceiv-ably, the stacking between residues in the T-loopand residues A130 and A180 (Fig. 6) might play arole in this process. Available data suggest that Mg2+

is bound to the substrate close to the phosphorous tobe attacked, and we have presented a model wherethis Mg2+ is responsible for generating the activatedH2O that acts as the nucleophile.2,51 The distancebetween this Mg2+ and the phosphorous is 6.7 Å(referred to as P(+1) in Fig. 6c), while the distance tothe phosphorous between position −1 and position−2 [P(−1)] is 7.27 Å (Fig. 6c).37 Therefore, failure tobreak the C−1/G+73 pair in the RPR substratecomplex18,29 alters the positioning of this Mg2+

such that cleavage occurs preferentially at −1.Changing the structure of the TBS region10,22 (thiswork) or replacing C at −1 with U22 facilitatesdisruption of C−1/G+73 (if present; the frequency ofthe C−1/G+73 pair in pre-tRNA can vary between0% and 21%, depending on bacteria29), therebydisfavoring or preventing an attack on P(−1) andfavoring an attack on P(+1). In this context, we notethat disruption of base-pairing in DNA has beendiscussed to result in binding of divalent metal ionsto additional sites.52 Moreover, the presence of a 2′H

at G+1 might result in a conformational change thatfacilitates disruption of C−1/G+73 and/or position-ing of the Mg2+ that activates the nucleophile H2O.In conclusion, this model emphasizes the interplaybetween separate regions upon formation of aproductive RPR–substrate complex that leads topositioning of the catalytic Mg2+.

Materials and Methods

Preparation of substrates and EcoRPR variants

All substrates were purchased from Dharmacon USAand purified on a 15% denaturing PAGE gel, followed byovernight Bio-Trap extraction and phenol–chloroformextraction. The different substrates were labeled with 32Por [γ-32P]ATP at their 5′ ends and gel purified usingstandard protocols, as previously described.22 The variousEcoRPRs were generated as runoff transcripts using T7DNA-dependent RNA polymerase and PCR-amplifiedtemplates, as described elsewhere.19,22,53

Assay conditions

The cleavage reactions were conducted in buffer C[50 mM 4-morpholineethanesulfonic acid and 0.8 MNH4Cl (pH 6.1)] at 37 °C in the presence of indicatedconcentrations of Mg(OAc)2. Before mixing preheated(37 °C) substrate with EcoRPR, we preincubated EcoRPR at37 °C in buffer C andMg(OAc)2 for at least 10 min to allowproper folding. In the Mg2+ titration experiments, theconcentration of substrates was 0.02 μM, while theconcentration of RPR was 0.8–1.6 μM (the concentrationvaried depending on the different substrate–EcoRPRcombinations). In the experiments where the cleavagerates were determined with one EcoRPR concentration(time-course experiments), the concentrations of sub-strates and EcoRPR were 0.02 μM and 4 μM, respectively.

Determination of the Hill coefficients

The Hill coefficients (n) for the cleavage of the differentsubstrates with the various RPRs were determined fromthe slopes of plots log[v/(Vmax−v)] versus log[Mg2+] (Kurzand Fierke54 and references therein).

Determination of kinetic constants undersingle-turnover conditions

The kinetic constants kobs and kobs/Ksto (=kcat/Km) were

determined under saturating single-turnover conditions inbuffer C at pH 6.1 and 800 mMMg(OAc)2. At this pH, thechemistry of cleavage is believed to be rate limiting, andkobs is referred to as the rate constant of cleavage (see themain text). The final concentration of the substrate was0.02 μM, and the concentration of EcoRPR ranged between0.1 μM and 51.2 μM, depending on the combination ofsubstrates and EcoRPRs. To calculate the kobs and kobs/K

sto

values, we determined the initial cleavage rates for at leastsix different RPR concentrations, where the lowest


concentration of RPR was at least five times higher thanthe concentration of the substrate. For the calculations, weused the 5′ cleavage fragments, and the time of cleavagewas adjusted to be within the linear part of the curve ofkinetics. The kobs and kobs/K

sto values were obtained bylinear regression from Eadie–Hofstee plots.Supplementary materials related to this article can be

found online at doi:10.1016/j.jmb.2011.05.049

Acknowledgements

We thank our colleagues for discussions andassistance. We thank Ms. T. Bergfors and Dr. S.Dasgupta for critical reading of the manuscript.Ongoing work is being funded by the SwedishResearch Council and Uppsala RNA Research Center(Linneaus support from the SwedishResearchCouncil)and in the past by the Foundation for StrategicResearch.

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