2007 Scott Curr Op Str Biol

COSTBI-455; NO OF PAGES 7 Ribozymes William G Scott The structural molecular biology of ribozymes took another great leap forward during the past two years. Before ribozymes were discovered in the early 1980s, all enzymes were thought to be proteins. No detailed structural information on ribozymes became available until 1994. Now, within the past two years, near atomic resolution crystal structures are available for almost all of the known ribozymes. The latest additions include ribonuclease P,
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  COSTBI-455; NO OF PAGES 7 Ribozymes William G Scott The structural molecular biology of ribozymes took anothergreatleap forwardduringthe pasttwoyears.Before ribozymeswere discovered in the early 1980s, all enzymes were thoughtto be proteins. No detailed structural information on ribozymesbecame available until 1994. Now, within the past two years,near atomic resolution crystal structures are available foralmost all of the known ribozymes. The latest additions includeribonuclease P, group I intron structures, the ribosome (thepeptidyl transferase appears to be a ribozyme) and severalsmaller ribozymes, including a Diels–Alderase, the glmS ribozyme and a new hammerhead ribozyme structure thatreconciles 12 years of discord. Although not all ribozymes aremetalloenzymes, acid-base catalysis appears to be a universalproperty shared by all ribozymes as well as many of theirprotein cousins.  Addresses Department of Chemistry and Biochemistry, and The Center for theMolecular Biology of RNA, 228 Sinsheimer Laboratories, University of California at Santa Cruz, Santa Cruz, CA 95064, USA Corresponding author: Scott, William G ( ) Current Opinion in Structural Biology  2007, 17 :1–7This review comes from a themed issue onNucleic acidsEdited by Dinshaw J Patel and Eric Westhof 0959-440X/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI10.1016/ Introduction Ribozymes are enzymes whose catalytic centers are com-posed entirely of RNA and therefore do not requireproteins for catalysis (although many exist naturally asRNA–protein complexes). Their discovery in the early1980s provided a potential logical escape from the con-undrum that plagues evolutionary molecular biology:which came first, information-encoding nucleic acids orproteins — including the enzymes needed to replicatenucleic acids? If RNA can do both, it is a potential self-replicase and the ‘RNA world’ hypothesis is based on theassumption that pre-biotic self-replicating moleculeswere composed of RNA.Perhaps the most important (apparent) ribozyme is theribosome, as the 23S RNAappears to comprisemost if notall of the active site [1,2];near atomic resolution struc- tures of the Escherichia coli  [3  ] and Thermus thermophilus [4  ,5  ] 70S ribosomes have now provided us with manynew structural insights. However, the main focus of thisreview will be to survey the many advances made inunderstanding the structural basis of ribozyme catalysis inother RNA systems. In reviewing these advances, itseems noteworthy that many of the most important onesemerged fromlaboratories that arenotprimarily knownasstructural biology research groups, but rather are labora-tories that focus primarily on the RNA molecules them-selves. Examples include Harry Noller’s ribosome group,Norm Pace’s RNase P group, Tom Cech’s intron groupandScottStrobel’sRNAchemistrygroup.Theblurringof such distinctions will probably be a growing trend in thefuture of macromolecular structural studies and is anencouraging development.All ribozymes were believed srcinally to be metalloen-zymes,requiringMg 2+ orotherdivalentmetalionsforbothfolding and catalysis. A ‘two-metal mechanism’ had beenproposed[6] inwhichhydratedMg 2+ ionsplayedtherolesofgeneralacidsandbases.Thispredictionappearstohavebeen correct for the group I intron (the specific context of thesrcinalproposal),ashasnowbeenrevealedbyStahleyand Strobel [7  ] (described below), but perhaps the moststriking result is that it does not generalize to all ribozymesystems. Acid-base catalysis appears to be a catalyticstrategy so fundamental that it occurs in both proteinand RNA enzymes; in many cases, it seems that theRNA itself, rather than acting as a passive scaffold formetal ion binding, is an active participant in acid-basecatalysis in the sense that nucleotide functional groups,rather than metal complexes, often mimic the roles thatamino acids play in the active sites of protein enzymes.Several of the small self-cleaving RNAs as a consequencedo not strictly require divalent metal ions for catalysis [8]and no divalent metal ions have yet been observed in theactive site of the peptidyl transferase, the ribozyme that isembedded in the ribosome. Ribonuclease P RibonucleaseP(RNaseP) wasthefirst trueRNA enzymeidentified [9]. An RNA–protein complex, the catalyticsubunit of bacterial RNase P is composed entirely of RNA (and it is thought that this is the case with theeukaryotic version as well). It processes precursor tRNAsand other RNAs required for cellular metabolism.Although structural fragments of RNase P have beenpreviously elucidated, two structures of the entire cata-lytic RNA subunit finally appeared simultaneously in2005, one from Thermotoga maritima at 3.85 A˚resolution[10  ] and the other from Bacillus stearothermophilus at3.3 A˚resolution [11  ]. Both ribozyme structures were Current Opinion in Structural Biology  2007, 17 :1–7 Please cite this article in press as: Scott WG, Ribozymes, Curr Opin Struct Biol (2007), doi:10.1016/  obtained in the absence of bound substrate and proteinnon-catalytic subunit, so active site interactions betweenenzyme and substrate can only be inferred. The B. stearothermophilus fold is depicted inFigure 1. A modelfor the structure of eukaryotic RNase P has also beenproposed based on these crystal structures [12]. Group I intron The group I intron was the first catalytic RNA discovered[13]and,in1986, itwas demonstratedtobeatrueenzyme[14]. Four group I intron crystal structures are nowavailable, two of which fall into the current review period.Following publication of the Azoarcus [15] and Tetrahy-mena [16] group I intron ribozyme structures in 2004, twoparticularly noteworthy structures have appeared. In Jan-uary 2005, the structure of aphage Twort  group I ribo-zyme–product complex [17  ] appeared (Figure 2a),followed in September by a second structure of the  Azoarcus group I intron ribozyme [7  ], which revealedevidence of a two-metal-ion mechanism in ribozymecatalysis (Figure 2b,c).The folds of the various group I introns are quite similar[18  ],permittingcomparisonsbetweenmolecularspecies.The first Azoarcus structure was in a pre-catalytic state, inwhich both exons (the substrate of the reaction in whichadjacent exons are spliced as the intron excises itself)were present. The Tetrahymena group I intron structurerepresents a state in which the 3 0 -terminal v -guanosineand a metal ion are present in the active site. The newerstructures complement these two states with an enzyme–product complex, and a complex in which all substrate,ribozyme functional groups and predicted metal ions arepresent in the active site. These are observed as a clusterof two metal ions, each of which is coordinated to no lessthan five phosphate or ribose oxygens in the active site.The Twort  ribozyme in addition reveals how the periph-eralinsertionsthatarecharacteristicofphageintronsformaringthatcompletelyenvelopstheactive site(Figure2a).  A Diels–Alderase ribozyme Possibly every crystallographer’s favorite reaction fromorganic chemistry is the Diels–Alder reaction, as it revealshow orbital symmetry dictates reactivity between a dieneand a dienophile. Proteins and RNAs have both beenevolved in vitro that enzymatically catalyze this reaction.It is remarkable that RNA, originally thought to beincapable of enzymatic catalysis and then thought tocatalyze primarily phosphodiester reactions, is capableof catalyzing carbon–carbon bond formation. The proteinDiels–Alderase is a catalytic antibody whose structure isknown. We now have the structure of a Diels–Alderribozyme in both the unbound and enzyme–productcomplex states (Figure 3a), revealing that the ribozymeuses a combination of proximity, spatial complementarityand electronic effects (Figure 3b) to activate stereoselec-tivecatalysis, reminiscent of the protein Diels–Alderase[19  ]. The glmS ribozyme The glmS  ribozymeis a recently discovered ribozyme thatis unique in the world of naturally occurring ribozymes intwo respects. First, it is a ribozyme that is also a ribos-witch. Second, the regulatory effector of the ribozyme,glucosamine-6-phosphate (GlcN6P), is actually a func-tional group that binds to the ribozyme active site andparticipates in the acid-base catalysis of RNA self-clea-vage. The glmS  ribozyme is derived from a self-cleavingRNA sequence found in the 5 0 -untranslated region (5 0 -UTR) of the glmS  message; it cleaves itself, inactivatingthe message, when the cofactor GlcN6P binds. GlcN6Pproduction is thus regulated in many Gram-positive bac-teria via this ribozyme-mediated negative-feedbackmechanism. 2 Nucleic acids COSTBI-455; NO OF PAGES 7 Figure 1 Stereo view of the fold of the RNase P RNA, as determined from B. stearothermophilus . The 5 0 end of the molecule is in blue and the 3 0 end is in red. Current Opinion in Structural Biology  2007, 17 :1–7 Please cite this article in press as: Scott WG, Ribozymes, Curr Opin Struct Biol (2007), doi:10.1016/  Ribozymes Scott 3 COSTBI-455; NO OF PAGES 7 Figure 2 Group I intron structures. (a) Cartoon representation of the fold of the Twort  group I intron. The 5 0 end is dark blue and the 3 0 end is red. Severalobserved Mg 2+ ions are depicted as grey spheres. (b) The previously reported Azoarcus group I intron structure (grey cartoon) shares manystructural features with the Twort  group I intron. The new Azoarcus structure reveals interactions between two Mg 2+ ions (cyan) at the activesite and the 5 0 end of the 3 0 intron (blue), the 3 0 end of the 5 0 intron (red) and the v -guanosine cofactor (green). (c) Close-up stereo view of theactive site in (b). Hydrogen bonds between the residues (including the v -guanosine) that comprise the cofactor-binding site are shown as lightblue dotted lines. Figure 3 Cartoon representation of the fold of the Diels–Alder ribozyme (a) . The 5 0 end is colored blue and the 3 0 end of the RNA is colored red, alongwith a space-filled representation of the bound adduct. (b) Close-up stereo view of the active site (same color scheme), depicting aromaticstacking and other interactions between the ribozyme and the Diels–Alder Current Opinion in Structural Biology  2007, 17 :1–7 Please cite this article in press as: Scott WG, Ribozymes, Curr Opin Struct Biol (2007), doi:10.1016/  The structure of the glmS  ribozyme is thus of particularinterest both as a riboswitch and as an unusual catalyticRNA. As it is known to occur only in Gram-positivebacteria, it is also a potential antibiotic target. The struc-ture of the glmS  ribozyme is discussed in the context of riboswitches in an accompanying review by Ferre´ -D’Amare´ and colleagues in this issue. Here, I focus onthe structure from the point of view of catalysis.Several structures of the glmS  ribozyme are now available.The first set, solved by Klein and Ferre´ -D’Amare´ [20  ],comprises four ribozyme structures derived from Thermo-anaerobacter tengcongensis ,including2 0 -NH 2 -and 2 0 -deoxy-modified cleavage site structures (PDB codes 2GCS and2H0S), an unmodified cleavage–product structure (PDBcode 2GCV) without cofactor bound and a complexstructure in which Gly6P, a highly homologous structuralanalogue of GlcN6P that is a competitive inhibitor of theribozyme, is bound to the 2 0 -deoxy-modified glmS  ribo-zyme (PDB code 2H0Z). An additional structure from  Bacillusanthracis , solved by Cochrane, Lipchock andStrobel [21  ], complements the srcinal structures, as itwas obtained with GlcN6P bound. Together, they pro-vide a fairly complete structural analysis of how a ribos-witch-ribozyme is activated.The fold of the glmS  ribozyme is that of a double pseu-doknot, as depicted inFigure 4a. The cofactor-bindingsite is positioned immediately adjacent to the scissilephosphate, as seen inFigure 4b, a close-up stereo viewin which several interactions between the RNA, thecofactor and the scissile phosphate are depicted. Becausethe inhibitor Gly6P differs from GlcN6P in only one non-hydrogen atom, it was proposed that most, if not all, of theinteractions seen in the complex structure are represen-tative of those with which the ribozyme binds its naturalcofactor, GlcN6P. The B. anthracis structure indeed vali-dates this claim. The differences are fairly subtle, butinclude the presence of a hydrogen bond to the C4-OH of the cofactor. The C2-NH 2 amine in GlcN6P and thecorresponding C2-OH in Gly6P are positioned withinhydrogen-bonding distance of the 5 0 -oxygen leavinggroup, together suggesting that GlcN6P is the generalacid catalytic component of the self-cleavage reaction.G40 (G33 in the B. anthracis structure) in turn is posi-tioned such that its N1 is within hydrogen-bonding dis-tance of the nucleophilic 2 0 -OH at the ribozyme cleavagesite, suggesting that G40 may be the general base com-ponent (similar to what is seen in the hammerheadribozyme structure, described below).Structures of the uncleaved RNA in the absence of thecofactor reveal that the substrate is positioned for in-lineattack in a pre-formed active site. Binding of the cofactorthen initiates the cleavage reaction by providing theacidic component to the catalyst. From a structuralperspective, it does not appear that any metal ions areinvolved directly in the chemistry of catalysis. 4 Nucleic acids COSTBI-455; NO OF PAGES 7 Figure 4 Cartoon representation of the fold of the glmS ribozyme (a) . The 5 0 end of each RNA strand is colored blue and the 3 0 end of each strand iscolored red, along with a space-filled representation of the bound cofactor. (b) Close-up stereo view of the active site (same color scheme),depicting the scissile phosphate as white atoms at the junction of the first two residues of the substrate strand (dark blue). Several stabilizinginteractions with the inhibitor cofactor Gly6P and the RNA and solvent are indicated with blue dotted lines. The active cofactor GlcN6Pdiffers from Gly6P in that the C2-OH is replaced with C2-NH 2 , making the cofactor a general acid catalyst that potentially supplies theleaving-group oxygen with a required proton. Current Opinion in Structural Biology  2007, 17 :1–7 Please cite this article in press as: Scott WG, Ribozymes, Curr Opin Struct Biol (2007), doi:10.1016/
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