N E W S A N D V I E W S

772 VOLUME 10 NUMBER 10 OCTOBER 2003 NATURE STRUCTURAL BIOLOGY

Using just four building blocks—A, C, G andU—RNA molecules adopt a remarkably widevariety of folds that allow them to play diverseroles throughout the process of gene expres-sion. Perhaps to augment the limited chemi-cal repertoire of RNA bases, species from allkingdoms of life have evolved a vast numberof enzymes that modify nucleobases aftertranscription. At last count, 96 different mod-ified nucleosides had been identified in RNA.It is becoming clear that base modifications,in particular, are functionally significant: theyare required for pre-mRNA splicing1; theyimprove translational fidelity2; and theyincrease RNA stability3. RNA-modifyingenzymes are expected to comprise up to∼ 10% of coding genomes4 and mutation inthe human pseudouridine synthase causesdyskeratosis congenita, a bone marrow–fail-ure disorder5.

The tRNA guanine transglycosylases(TGTs) have attracted attention because oftheir apparently unique ability to remove thetarget base and attach the modified basewithin a single active site. To facilitate thisdual activity, TGTs form an enzyme-tRNAcovalent intermediate. Bacterial and eukary-otic TGTs recognize and modify the wobblebase in the anticodon loop (position 34). Toidentify the nucleophile and determine thestructural basis of target-site selection of theseTGTs, Xie et al.6 report, on page 781 of thisissue of Nature Structural Biology, the firstviews of a covalent reaction intermediate anda product complex of a bacterial TGT. Theenzyme recognizes the backbone of the stem

of the anticodon stem-loop, and its base-binding pockets contact positions 34 and 35in a sequence-specific manner. Direct baserecognition is enabled by base flipping of thetarget and flanking bases. Asp280 is shown tobe the nucleophile, forming a bond to tRNAduring catalysis. This result was a surprisebecause mutational studies and active sitelocation suggested that a different aspartate,Asp102, was the nucleophile7,8. Like Asp102,Asp280 is invariant, essential for activity9 andresides in the active site. Future experimentsare needed to determine whether Asp102assists Asp280 by serving as a general base inthe reaction mechanism, as proposed by Xieet al.6 (Fig. 1).

The authors devised a clever method toreversibly trap the covalent intermediate and

to generate the product complex (Fig. 1). Totrap the covalent intermediate, the TGTenzyme was mixed with RNA substrate andexcess base analog (9-deazaguanine). The carboxyl group of Asp280 attacks the C1′atom of the target base, liberating the wobbleguanine and forming the enzyme-tRNA cova-lent intermediate. The excess 9-deazaguanineexchanges with the guanine in the base-binding pocket by mass action. A carbon atomat the 9 position renders the analog unreactivefor the next step of the reaction, thereby trap-ping the covalent intermediate that was subse-quently purified and crystallized. To generatethe product, modified base is added to crystalscontaining the trapped covalent intermediate.Fortuitously, initial binding of the modifiedbase is favored owing to additional stabilizing

The author is in the Department of Bio-chemistry and Molecular Biology, the Universityof Chicago, Chicago, Illinois 60637, USA. e-mail: ccorrell@uchicago.edu

Caught in the act of modifying tRNACarl C Correll

Recent studies of a tRNA guanine transglycosylase—a modifying enzyme known in all kingdoms of life—show how anunexpected active site aspartate becomes covalently attached to the tRNA substrate during catalysis, and how subsequent baseexchange occurs in a single active site.

HB

:B (Asp102?)

preQ1 G

9-deazaG

G preQ1

9-deazaG

HB

Initial Michaelis complex Covalent intermediate Product complex

Trapped covalent intermediate

Base exchange

Figure 1 Reaction scheme for bacterial tRNA guanine transglycosylase. The base-binding pocket formssequence-specific contacts to the guanine base, the modified base (blue) and the base analog (red).PreQ1 is a precursor to the modified base queuine. R and R′ represent tRNA sequences that flank thetarget nucleotide. Xie et al.6 determined crystal structures of both boxed reaction complexes.

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N E W S A N D V I E W S

In the ∼ 50 years since the formulation of thesliding-filament model of muscle contraction,intense investigation using tools of biochem-istry, biophysics and molecular biology has ledto a detailed understanding of many aspects ofactomyosin-based motility. The discovery ofcross-bridges (heads of the myosin molecule)and their conformational changes in the1960s, together with the elucidation of thekinetic mechanism of actomyosin ATPase inthe 1970s, led to a physical model of the cross-bridge cycle where the determined kinetic andthermodynamic parameters could be fit in alogical fashion (Fig. 1). Although the model ofthe cycle was stimulating, even at low resolu-tion, refinement and extension of the modeltoward the atomic level, which is essential for arigorous understanding of the mechanism ofenergy transduction, necessarily awaited high-resolution structure determination of themost important components, beginning withthe structure of actin in 1990 (ref. 1) andmyosin in 1993 (ref. 2).

Structural studies have revealed someimportant features that correlate with thedifferent nucleotide-bound states of themyosin head. In particular, the position ofthe switch II region of the myosin head, aswell as the angle of the lever arm region ofmyosin (an extended α-helix stabilized byinteraction with the light chains) relative tothe motor domain (which contains the ATP-and actin-binding sites) (reviewed in ref. 3),display structural changes that depend onthe occupancy and the nature of thenucleotides. Switch II exists mainly in twostates: in the presence of ATP, it is ‘closed,’and the glycine of the DXXG motif (con-served in many ATPases and GTPases)directly interacts with the γ-phosphate ofATP. In all other nucleotide states observedthus far, it is ‘open,’ and switch II has movedsignificantly away from the position of the γ-phosphate. Movement of switch II seemsto be coupled to a long-range conforma-tional change in myosin that leads to achange of the angle between the motordomain and the lever arm. This angle changeis thought to represent the power stroke inthe contractile mechanism, and its couplingto the loss of the γ-phosphate of ATP is considered to reflect the fundamental rela-

tionship between overall structure andnucleotide state in this system.

The description above paints only part ofthe picture, because it lacks another equallyimportant feature in the cross-bridge cycle,the coupling of nucleotide state to actin-binding affinity—that is, myosin–ATP andmyosin–ADP-Pi bind weakly to actin fila-ment, whereas myosin–ADP and myosinalone bind strongly. The transitionsbetween weakly and strongly bound statesof myosin represent the fundamental ther-modynamic event in the cross-bridge cycleleading to production of mechanical work4.Here, a direct link between the structuralstates of the actin- and nucleotide-bindingsites is crucial for further understanding themechanism of force generation. Such a linkhas not been observed until recently, leavingthese two most significant aspects of thecross-bridge cycle—change of lever armangle and change of actin affinity—concep-tually uncoupled. The publication of twoarticles in this issue of Nature StructuralBiology5,6 and two related reports inNature7,8 has changed this situation: thesestudies now take us an important step closerto understanding the actomyosin-basedmotility at atomic level.

NATURE STRUCTURAL BIOLOGY VOLUME 10 NUMBER 10 OCTOBER 2003 773

contacts from the exocyclic C7 moiety. Afterbase exchange, product is formed in situ,demonstrating that the enzyme is catalyticallyactive in the crystalline milieu.

Recent structural studies of TGTs andpseudouridine synthases suggest that thesemodifying enzymes share common substraterecognition and catalytic strategies3,10. Bothenzyme classes use base flipping to gain accessto their target sites. Shape recognition is usedto locate the target site, which is recognized bysequence-specific contacts in a base-bindingpocket. The observed RNA conformationalrearrangements are more extensive than thoseseen for DNA-modifying enzymes, wheretypically only the target base is flipped out ofthe stacked helix. To recognize their substrate,RNA-modifying enzymes unstack severalbases and rearrange the phosphodiester back-bone. Archaeal TGTs, unlike those from bac-

teria and eukarya, target a buried site (posi-tion 15) in the D-loop, where extreme sub-strate rearrangements are observed3. Thisarchaeal TGT pries open all the base pairs inthe D-loop, separating it from the T-loop, togain access to the target site. In contrast tothese significant rearrangements in the RNAstructure, only small adaptations areobserved in the TGT enzyme structures; addi-tional structural studies are needed to deter-mine the extent to which pseudouridinesynthases change conformation upon sub-strate binding.

Like TGTs, pseudouridine synthases areproposed to form a covalent intermediate viaan aspartate11. To catalyze isomerization ofspecific uridines, two competing mechanismshave been proposed: the nucleophile eitherattacks C6 of the uridine base or C1′ of theuridine sugar. After proper base rotation, the

uridine is re-attached to form a pseudouri-dine at the target site. Hopefully, novel trap-ping experiments will resolve this mechanisticdilemma by direct visualization of the cova-lent intermediate.

1. Yu, Y.T., Shu, M.D. & Steitz, J.A. EMBO J. 17,5783–5795 (1998).

2. Urbonavicius, J., Qian, Q., Durand, J.M., Hagervall,T.G. & Bjork, G.R. EMBO J. 20, 4863–4873 (2001).

3. Ishitani, R. et al. Cell 113, 383–394 (2003).4. Anantharaman, V., Koonin, E.V. & Aravind, L. Nucleic

Acids Res. 30, 1427–1464 (2002).5. Heiss, N.S. et al. Nat. Genet. 19, 32–38 (1998).6. Xie, W., Liu, X. & Huang, H.R. Nat. Struct. Biol. 10,

781–788 (2003).7. Romier, C., Reuter, K., Suck, D. & Ficner, R.

Biochemistry 35, 15734–15739 (1996).8. Romier, C., Reuter, K., Suck, D. & Ficner, R. EMBO J.

15, 2850–2857 (1996).9. Kittendorf, J.D., Sgraja, T., Reuter, K., Klebe, G. &

Garcia, G.A. J. Biol. Chem. JBC online, 8 August2003 (doi:10.1074/jbc.M304323200).

10. Hoang, C. & Ferre-D’Amare, A.R. Cell 107, 929–939(2001).

11. Mueller, E.G. Nat. Struct. Biol. 9, 320–322 (2002).

The missing link in the muscle cross-bridge cycleRoger S Goody

Four recent studies demonstrate that closing of a cleft in the myosin head is required for actin binding and nucleotide release.This represents a major breakthrough in understanding actomyosin-based motility.

The author is in the Department of PhysicalBiochemistry, Max-Planck-Institute ofMolecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.e-mail: goody@mpi-dortmund.mpg.de

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