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coherence effects are not deployed in thenormal operation of the best-characterizedphotosynthetic antenna, the light-harvestingcomplex 2 of the bacterium Rhodopseudo-monas acidophila. But they could play a partin a system with more extensive delocaliza-tion among the chromophores or at very low temperature.

The work of Jang et al.1 of course leavesplenty of scope for further investigations.

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For example, what is the interplay betweenthe length and time scales over which coher-ent nuclear fluctuations of chromophoresand their surrounding protein are pre-served? Can experiments be designed toreveal these quantum effects? And on thepractical side, how can these factors lead todesign strategies for synthetic light har-vesters with enhanced energy-transfer ratesand efficiencies? ■

Graham R. Fleming is in the Department ofChemistry, University of California, Berkeley,and at the Lawrence Berkeley National Laboratory,Berkeley, California 94720, USA.e-mail: grfleming@lbl.govGregory D. Scholes is in the Department ofChemistry, University of Toronto, Lash MillerChemical Laboratories, 80 St George Street, Toronto,Ontario M5S 3H6, Canada.e-mail: gscholes@chem.utoronto.ca1. Jang, S., Newton, M. D. & Silbey, R. J. Phys. Rev. Lett. 92,

218301 (2004).

2. Sundström, V., Pullerits, T. & van Grondelle, R. J. Phys. Chem. B

103, 2327–2346 (1999).

3. Förster, Th. Ann. Phys. 2, 55–75 (1948).

4. Van Der Meer, B. W., Coker, G. & Chen, S.-Y. S. Resonance

Energy Transfer: Theory and Data (VCH, New York, 1994).

5. Scholes, G. D., Jordanides, X. J. & Fleming, G. R. J. Phys. Chem.

B 105, 1640–1651 (2001).

6. van Amerongen, H., Valkunas, L. & van Grondelle, R.

Photosynthetic Excitons (World Scientific, Singapore, 2000).

7. Sumi, H. J. J. Phys. Chem. B 103, 252–260 (1999).

8. Scholes, G. D. & Fleming, G. R. J. Phys. Chem. B 104, 1853–1868

(2000).

9. Ambrose, W. P. & Moerner, W. E. Nature 349, 225–227 (1991).

10.Holt, N. E., Fleming, G. R. & Niyogi, K. K. Biochemistry 43,

8281–8289 (2004).

11. Jordanides, X. J., Scholes, G. D., Shapley, W. A., Reimers, J. R. &

Fleming G. R. J. Phys. Chem. B 108, 1753–1765 (2004).

systems with widely spaced chromophoresmay be crucial in increasing energy-transferefficiency.

Typically,a confined multichromophoricsystem allows energy to be moved morerapidly and effectively using a broad windowof acceptor states.As a result,new definitionsare needed to characterize the donor and the acceptor. Physically, they each consist ofmultiple chromophores; spectroscopically,they behave as electronically delocalizedstates. The properties of these states arestrongly modified by interactions with the chromophores’ environments, makingeach donor–acceptor pair in the ensembleunique.That concept is familiar from single-molecule experiments9, where the spectro-scopy of an individual molecule is influencedby its local environment. A general conse-quence is that absorption and fluorescencemeasurements cannot be used to record ameaningful measure of the energy transferrate, as in the spectral overlaps that consti-tute the backbone of Förster theory.

These realizations led to the developmentof generalized Förster theory, which hashelped to resolve several long-standing mysteries in photosynthesis. Paradoxically,for instance, it has emerged that the sameinteractions that produce perfectly efficientenergy transport also allow photosyntheticorganisms to construct molecular safetyvalves that dissipate excess excitation energythat would otherwise cause irreversibledamage10,11. Furthermore, this work hasshown how photosynthetic systems exploitenergetic disorder to improve spectral coverage, and reduce energy mismatches tomake the system exceedingly robust against thermal and structural variations.

Jang et al.1 have further explored themulti-component donor–acceptor aspect ofnatural antennae, and report an improvedmethod of calculating the spectral overlapfactors in the generalized Förster theory for predicting the rate of energy transfer incomplex multichromophoric systems. Theyconsider an additional ingredient: a kind ofinterchromophore term that accounts forquantum mechanical coherence among thechromophores that collectively donate, oraccept,excitation energy.Such an effect,if notdestroyed by environmental fluctuations ontimes shorter than that on which the energytransfer occurs, could lead to increased ratesof energy flow. However, it is still assumedthat such a quantum effect is insignificant inthe donor–acceptor interaction.

In the context of this theory, we can nowdetermine whether coherent nuclear fluctu-ations throughout a protein or syntheticstructure can be used to accelerate the rate of energy transfer. A signature of coherenceeffects is that the entity acting as a donor oran acceptor will effectively change size as theamplitude of the environmental fluctuationschanges with a change in temperature. Such

Molecular biology

Genetic code seizes pyrrolysinePaul Schimmel and Kirk Beebe

Identification of the enzyme that mediates insertion of a rare aminoacid, pyrrolysine, into protein solves a puzzle and expands the rules of the genetic code established nearly half a century ago.

The discovery of the genetic code thattranslates the information containedin DNA into proteins ranks as one of

the greatest achievements of the past century.The code appeared before the split of the treeof life into the three great kingdoms — Bac-teria, Archaea and Eukarya — and has beenmaintained in all organisms as an algorithmthat relates nucleotide triplets in genes tospecific amino acids in proteins. The coderequires for its implementation a set of 20universal enzymes — aminoacyl tRNA synthetases (one for each amino acid). Buton page 333 of this issue, Blight et al.1 showthat, in certain archaea, an unusual, naturalamino acid is read by the code using a noveltwenty-first aminoacyl tRNA synthetase.

The adaptors responsible for translatingthe genetic code from the messenger RNA(mRNA) into amino-acid sequence are the

transfer RNAs (tRNAs). Each tRNA bears an‘anti-codon’ sequence that corresponds tothe nucleotide triplet code for a specificamino acid. Each of the canonical 20 aminoacids is covalently linked to the tRNA thatbears the anti-codon corresponding to theparticular amino acid. This charging reac-tion, an aminoacylation reaction, is carriedout by the aminoacyl tRNA synthetases,eachsynthetase being specific for a particularamino acid and tRNA. Essentially, the tRNAreads the position in the mRNA sequencewhere its cognate amino acid is supposed toappear, and holds the amino acid in positionuntil it is incorporated into the growing peptide chain. Three nucleotide triplets —UAA, UAG and UGA — are used as stop signals.

Much effort has gone into artificially creating (typically by mutagenesis) new

Figure 2 Rhodopseudomonas — a bacterium that makes light work.

A.P

ASI

EK

A/S

PL

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aminoacyl tRNA synthetase–tRNA pairsthat could, in turn, insert novel amino acidsinto polypeptides, to create proteins withnovel properties2. The usual approachrequires engineering a tRNA to have an anti-codon that corresponds to a stop codon, sothat instead of signalling a stop, the tRNAwill insert an artificial amino acid. Butalthough these studies demonstrate thepower of artificial synthetase–tRNA pairs forintroducing novel amino acids into proteins,no natural examples were known to exist.

The novelty of the work by Blight et al.1

rests on the identification of a natural twenty-first synthetase that directly activates pyrro-lysine (both in vivo and in vitro). Pyrrolysine(pLys) is a non-canonical amino acid that isfound in certain methyltransferase enzymesof archaea3,4, where it is incorporated at aUAG codon, instead of there being a stop.The typical tRNA-charging reaction occursin two steps: activation of the amino acidwith adenosine triphosphate (ATP) to forman aminoacyl adenylate, and reaction of theadenylate with the tRNA to link the two.Blight et al.1 show that pyrrolysine tRNA synthetase activated pyrrolysine (to formpyrrolysyl-AMP) in vitro, and did not acti-vate any of the 20 canonical amino acids,including lysine. They also demonstrate that, both in vitro and in vivo, the enzymecatalyses the overall aminoacylation reac-tion, requiring a pyrrolysine-specific tRNA(tRNApLys), which has an anti-codon that iscomplementary to UAG.Recently,Polycarpoet al.5 reported similar findings in vitro (butnot in vivo). Thus, a natural synthetase–tRNA pair brings a new amino acid into thegenetic code.

Next, the authors1 expressed the pyrro-lysine-specific synthetase and tRNApLys in the bacterium Escherichia coli together withmethyltransferase from the archaean Metha-nosarcina barkeri, a protein that naturallycontains pyrrolysine. (Neither the two pro-teins nor tRNApLys are naturally present in E. coli.) Remarkably, when pyrrolysine wasadded to the culture, it was incorporated into the methyltransferase at the expectedposition. Thus, all the information requiredto expand the genetic code is contained intRNApLys, the pyrrolysine-specific synthetaseand the mRNA encoding the M. barkerimethyltransferase. No other componentsfrom the archaea are needed for the reactionto work in E. coli.

Another surprise from Blight and col-leagues’ work is that insertion of pyrrolysineinto the growing methyltransferase poly-peptide in vivo is highly efficient (greater than75% efficiency),much more so than is usuallyseen with the artificial tRNA–synthetasepairs that act at the UAG codon (typically less than 20% efficiency)6. (The authors donot report any experiment to test whethertRNApLys can also insert pyrrolysine at an artificially introduced UAG codon of a non-

methyltransferase mRNA.) However, weretRNApLys to act efficiently at the many natu-rally occurring UAG stop codons in E.coli, theresult would be expected to be toxic (whichwas not seen). This implies that tRNApLys cansomehow ‘find’specific UAG sequences.

We imagine that the local mRNA struc-ture around the target UAG is arranged in away that tRNApLys can recognize. A possibleprecedent is the mechanism by whichselenocysteine is inserted into proteins. Thisrare amino acid is formed by a modificationof the canonical amino acid serine while it is attached to a special tRNA7. (By analogy,an early proposal for how pyrrolysine wasinserted involved a similar modification oflysine4,8.) In bacteria, the selenocysteine-encoding UGA codon is targeted by a specialelongation factor, SelB, that binds specifi-cally to the selenocysteine-loaded tRNA.SelB also binds to a special RNA hairpin loop(the ‘selenocysteine insertion element’) thatsignals where the selenocysteine should go7 (Fig.1a).

How does the pyrrolysine-loadedtRNApLys identify the pyrrolysine-specificUAG codons in methyltransferase RNAs?Interestingly, RNA hairpin loops similar tothe selenocysteine insertion elements are predicted to lie near the pyrrolysine-encod-ing UAG codons9 (Fig. 1b). But with no other factors from methanogenic archaeaexpressed in E. coli, the possibility of a SelB-like component in E. coli has to be consid-ered. Could that factor be SelB itself ? Theproblem is that eukaryotes and probablyarchaea require two factors for seleno-cysteine insertion: a SelB-like elongation factor, and a second factor for binding theselenocysteine insertion element that has no

counterpart in E. coli 7,10. Could pyrrolysine-specific synthetase have a role in the selectionof not only tRNApLys, but also the pyrrolysineinsertion site, perhaps not releasing pyrro-lysine-loaded tRNA until it has reached thecorrect position?

Although many questions remain, themain finding is that the non-canonical aminoacid pyrrolysine can be translated by thegenetic code using a natural synthetase–tRNA pair. In some ways, this discovery reaffirms that notion that, if something canbe done by experiment, it has probably beendone earlier by nature. As an example, thedemonstration that RNAs could be experi-mentally selected to bind specific small mol-ecules foretold the eventual discovery of suchRNA ‘aptamers’ in bacteria11. With genomesand proteomes of so many species yet to beanalysed, one wonders what other surprisesthe genetic code has in store. ■

Paul Schimmel and Kirk Beebe are at The SkaggsInstitute for Chemical Biology, The Scripps ResearchInstitute, Beckman Center, BCC379, 10550 NorthTorrey Pines Road, La Jolla, California 92037, USA.e-mail: schimmel@scripps.edu1. Blight, S. K. et al. Nature 431, 333–335 (2004).

2. Thorson, J. S. et al. Methods Mol. Biol. 77, 43–73 (1998).

3. Hao, B. et al. Science 296, 1462–1466 (2002).

4. Srinivasan, G., James, C. M. & Krzycki, J. A. Science 296,1459–1462 (2002).

5. Polycarpo, C. et al. Proc. Natl Acad. Sci. USA 101, 12450–12454

(2004).

6. Anderson, J. C. & Schultz, P. G. Biochemistry 42, 9598–9608

(2003).

7. Hatfield, D. L. & Gladyshev, V. N. Mol. Cell. Biol. 22, 3565–3576

(2002).

8. Polycarpo, C. et al. Mol. Cell 12, 287–294 (2003).

9. Namy, O., Rousset, J. P., Napthine, S. & Brierley, I. Mol. Cell 13,157–168 (2004).

10.Rother, M., Wilting, R., Commans, S. & Böck, A. J. Mol. Biol.

299, 351–358 (2000).

11.Mandal, M. & Breaker, R. R. Nature Rev. Mol. Cell Biol. 5,451–463 (2004).

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STOP STOPUGA3′ 3′5′ 5′

Selenocysteine (SECIS) Pyrrolysine (PYLIS)

UG

A

STOPUAG (N5–6) 3′5′

a bBacteria Eukaryotes and Archaea

Archaea

Figure 1 RNA structures signal incorporation of non-canonical amino acids. a, A hairpin structurecalled the ‘selenocysteine insertion element’ (SECIS) is present in the messenger RNA thatcorresponds to where selenocysteine is due to be inserted into the growing peptide chain. In bacteria, SelB binds to tRNA loaded with selenocysteine and to SECIS, so that instead of UGAsignalling a stop, selenocysteine is added into the peptide. (In eukaryotes and archaea, two proteins perform the SelB function.) b, Predicted pyrrolysine insertion elements (PYLIS) in themRNA encoding archaeal methyltransferase. These are proposed to signal for pyrrolysine to beinserted in the methyltransferase peptide at the UAG codon (also usually a stop codon) in a mannersimilar to SECIS. Structures adapted from Namy et al.9.

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