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The Junction the Folding of the Hairpin Ribozyme Downloaded from www.rnajournal.org on February 14, 2006 The folding of the hairpin ribozyme: dependence on the loops and the junction Z. Y. Zhao, T. J. Wilson, K. Maxwell and D. M. Lilley RNA 2000 6: 1833-1846 References Article cited in: http://www.rnajournal.org/cgi/content/abstract/6/12/1833#otherarticles Email alerting Receive free email alerts when new articles cite this article - sign up in the box at the service top right corner of the article or click here Notes To subscribe to RNA go to: http://www.rnajournal.org/subscriptions/ © 2000 RNA Society Downloaded from www.rnajournal.org on February 14, 2006 RNA (2000), 6:1833–1846+ Cambridge University Press+ Printed in the USA+ Copyright © 2000 RNA Society+ The folding of the hairpin ribozyme: Dependence on the loops and the junction ZHENG-YUN ZHAO, TIMOTHY J. WILSON, KAERA MAXWELL, and DAVID M.J. LILLEY CRC Nucleic Acid Structure Research Group, Department of Biochemistry, The University of Dundee, Dundee DD1 4HN, United Kingdom ABSTRACT In its natural context, the hairpin ribozyme is constructed around a four-way helical junction. This presents the two loops that interact to form the active site on adjacent arms, requiring rotation into an antiparallel structure to bring them into proximity. In the present study we have compared the folding of this form of the ribozyme and subspecies lacking either the loops or the helical junction using fluorescence resonance energy transfer. The complete ribozyme as a four-way junction folds into an antiparallel structure by the cooperative binding of magnesium ions, requiring 21 20–40 mM for half-maximal extent of folding ([Mg ]1/2) and a Hill coefficient n 5 2. The isolated junction (lacking the loops) also folds into a corresponding antiparallel structure, but does so noncooperatively (n 5 1) at a higher 21 magnesium ion concentration ([Mg ]1/2 5 3 mM). Introduction of a G 1 1A mutation into loop A of the ribozyme results in a species with very similar folding to the simple junction, and complete loss of ribozyme activity. Removal of the junction from the ribozyme, replacing it either with a strand break (serving as a hinge) or a GC5 bulge, results in 21 greatly impaired folding, with [Mg ]1/2 . 20 mM. The results indicate that the natural form of the ribozyme undergoes ion-induced folding by the cooperative formation of an antiparallel junction and loop–loop interaction to generate the active form of the ribozyme. The four-way junction thus provides a scaffold in the natural RNA that facilitates the folding of the ribozyme into the active form. Keywords: FRET; RNA folding; RNA catalysis INTRODUCTION ceeds with inversion of configuration at the phosphorus (van Tol et al+, 1990; Koizumi & Ohtsuka, 1991)+ This RNA conformation and function are tightly linked+ Cat- indicates that the cleavage reaction occurs by an at- alytic RNA molecules (ribozymes) must undergo fold- tack of the oxygen atom of the 29-hydroxyl group on the ing into their correct three-dimensional structure to be 39 phosphorus atom by an S 2 mechanism+ It is prob- catalytically active, and mutations that cause aberrant N able that a part of the origin of catalysis lies in a local folding lead to loss of activity+ There is a very important distortion of RNA structure to facilitate the correct jux- electrostatic component in the folding process of RNA, taposition of attacking nucleophile and leaving group in arising from the highly charged phosphodiester back- the transition state of the reaction+ In the context of the bone, and thus metal ions play a critical role in stabi- local environment of the ribozyme active site, this leads lizing the folded structures+ to accelerated cleavage+ The linkage between folding and function is well il- There are a number of nucleolytic ribozymes that lustrated in the small nucleolytic ribozymes (Lilley, 1999)+ appear to proceed via essentially the same chemical These are relatively small RNA species that catalyze mechanism+ These include the hammerhead (Hutchins site-specific cleavage of the RNA backbone+ The prod- et al+, 1986; Forster & Symons, 1987; Uhlenbeck, 1987; ucts of the cleavage reaction are 59-hydroxyl and 29,39- Hazeloff & Gerlach, 1988), hairpin (Buzayan et al+, 1986; cyclic phosphate termini (Buzayan et al+, 1986; Hutchins Feldstein et al+, 1989; Hampel & Tritz, 1989), hepatitis et al+, 1986; Uhlenbeck, 1987), and the reaction pro- delta virus (Sharmeen et al+, 1988; Wu et al+, 1989) and Neurospora VS (Saville & Collins, 1990) ribozymes+ Reprint requests to: David M+J+ Lilley, CRC Nucleic Acid Structure The hairpin ribozyme (Fedor, 2000) occurs in the neg- Research Group, Department of Biochemistry, The University of Dundee, Dundee DD1 4HN, United Kingdom; e-mail: dmjlilley@ ative strand of the satellite RNA of the tobacco ringspot bad+dundee+ac+uk+ virus (Buzayan et al+, 1986; Feldstein et al+, 1989; 1833 Downloaded from www.rnajournal.org on February 14, 2006 1834 Z.-Y. Zhao et al. Hampel & Tritz, 1989) and similar plant viruses cleavage activity consists of the two duplexes contain- (DeYoung et al+, 1995), where it is required for the gen- ing the A- and B-loops, hinged at the continuous strand eration of the unit-sized 359-nt satellite RNA molecules that connects them+ Cleavage occurs in the A-loop following rolling circle replication+ The motif catalyzes (Fig+ 1A, arrow)+ Most of the bases and functional groups self-cleavage in the presence of magnesium ions, to- that are essential for catalytic activity lie in the two gether with the reverse ligation reaction (Buzayan et al+, loops (Berzal-Herranz et al+, 1993; Chowrira et al+, 1993; 1986), leading to an equilibrium between cleavage and Anderson et al+, 1994; Grasby et al+, 1995; Schmidt ligation (Hegg & Fedor, 1995)+ et al+, 1996; Siwkowski et al+, 1997; Shippy et al+, 1998; Figure 1A shows the proposed secondary structure Ryder & Strobel, 1999; Young et al+, 1999), and it was of the tobacco ringspot virus satellite RNA in the region therefore anticipated that these loops must interact to of the hairpin ribozyme+ The minimal form that retains generate the active form of the ribozyme+ This was FIGURE 1. The hairpin ribozyme in its natural context as a four-way helical junction+ A: Schematic showing the local secondary structure of the tobacco ringspot virus satellite (2) RNA in the region of the hairpin ribozyme+ The essential elements for RNA catalysis are the two loops, A and B, and ribozyme cleavage occurs at the position indicated by the arrow+ B: The base sequence of and around the hairpin ribozyme (Hampel & Tritz, 1989)+ The four arms of the junction are labeled A through D in a circular manner, where arms A and B contain the A and B loops, respectively+ The strands are designated with lower case letters, taken from the name of the arm in which the 59-terminus of the strand is located+ C: The hairpin ribozyme drawn in an antiparallel orientation+ This conformer is formed by coaxial stacking of arms B on C and A on D, whereupon b and d are continuous strands whereas strands a and c exchange between helical axes+ D: An example of a fluorophore-labeled hairpin ribozyme species used in FRET analysis of global structure+ The vectors are labeled according to the arms carrying donor (fluorescein) and acceptor (Cy3) in that order+ The terminal helix of arm B is 2 bp longer than in our previous studies (Murchie et al+, 1998; Walter et al+, 1998c)+ E: The sequence of loop A, showing the numbering used in the text+ The 29 modifications are introduced at A 2 1, and the sequence variation at G 1 1+ Downloaded from www.rnajournal.org on February 14, 2006 Folding of the hairpin ribozyme 1835 generally indicated by experiments in which the loop- cooperatively on binding divalent metal ions, and the containing duplexes were connected in a variety of dif- junction must be regarded as an integral functional com- ferent ways with retention of activity (Komatsu et al+, ponent of the ribozyme+ 1994, 1996, 1997a, 1997b, Butcher et al+, 1995; Shin et al+, 1996; Earnshaw et al+, 1997)+ Close physical proximity of the ends of the arms containing the loops RESULTS has been demonstrated by fluorescence resonance en- ergy transfer (FRET) (Murchie et al+, 1998; Walter et al+, Analysis of the global folding of the hairpin 1998d)+ NMR structures have been derived for the A- ribozyme using FRET , +, (Cai & Tinoco 1996) and B- (Butcher et al 1999) loops We have used FRET for the analysis of the global struc- , in isolation and models have been proposed for the ture of the hairpin ribozyme in its four-way junction manner of the interaction between the two loops via form (Murchie et al+, 1998; Walter et al+, 1998c), and +, , backbone (Earnshaw et al 1997 2000) and base (Pi- the isolated junction derived from it by removal of the A +, + nard et al 1999) contacts and B loops (Walter et al+, 1998b)+ In this approach we Examination of the secondary structure shows that compare the relative lengths of the six possible end- , in the natural context in the satellite RNA the loop- to-end vectors by measuring the efficiency of energy containing arms are connected not by a flexible hinge transfer (E ) between donor–acceptor fluorophore + FRET but as four arms of a perfect four-way helical junction pairs attached at pairs of 59-termini; in these experi- + The sequence around the junction is shown in Figure 1B ments we have used fluorescein and cyanine-3 (Cy-3) Four-way RNA junctions fold by pairwise coaxial stack- as a donor–acceptor pair+ The fluorescent species are , ing under all conditions into
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