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Millennium issue Constructing an RNA world David P. Bartel and Peter J. Unrau

A popular theory of lifeÕs origins states that the first biocatalysts were not made of but were made of RNA or a very similar polymer. Experiments are beginning to confirm that the catalytic abilities of RNA are compatible with this ÔRNA worldÕ hypothesis. For example, RNA can synthesize short fragments of RNA in a template-directed fashion and promote formation of , ester and glycosidic linkages. However, no known activity fully represents one presumed by the ÔRNA worldÕ theory, and reactions such as oxidation and reduction have yet to be demonstrated. Filling these gaps would place the hypothesis on much firmer ground and provide components for building minimal forms of RNA-based cellular .

ince the of Darwin and his ‘warm little pond’, scientists easily forgotten. Problems remain, particularly the implausibility have been wrestling with the challenge of formulating a of prebiotic RNA synthesis and stability8–10. Indeed, most profes- S plausible scenario for the origin and early of life. sional advocates of an RNA world are doubtful that life began How did a self-replicating assembly of emerge on the with RNA per se. Instead, they propose that life began with an early and give rise to cellular life? From this perspective, RNA-like polymer, yet to be identified, that possessed the cata- even the simplest free-living in contemporary are lytic and templating features of RNA but miraculously lacked of staggering complexity; Aquifex aeolicus, a simple autotrophic RNA’s undesirable traits, most notably, its intractable prebiotic bacterium, has 1512 open reading frames and at least 35 RNA synthesis4,8–11. The era of this RNA-like polymer is the ‘pre-RNA genes1. Because hundreds of these are common to all branches world’, which presumably gave rise to the RNA world in a manner of life, phylogenetic comparison cannot whittle away the analogous to that in which the RNA world gave rise to the of cellular life to a tractable set of genes representing the first . protein–nucleic-acid world of today. Relegating the RNA world The record for life appears to go back 3.5–3.85 billion years to a crucial intermediate in the early evolution of life rescues the – nearly as far back as the oldest terrestrial rocks2,3. Yet some of hypothesis from the ridicule of prebiotic but still does the oldest resemble modern , providing few not place it on firm footing. The ‘RNA world’ scenario hinges on clues as to how the earliest life differed from that of today. some rather far-fetched assumptions about the catalytic capability Because approaching the origin of life by looking back through of RNA. For example, RNA must have been phylogenetic or palaeontologic evidence has proved difficult, one responsible for replicating the ribozymes of the RNA world, includ- might hope to work forward instead, starting with the molecules and ing themselves (via their complementary sequences). RNA repli- conditions thought to prevail on the early Earth. However, only a cation is a very challenging set of reactions – far more challenging minute fraction of the possible combinations of molecules, con- than those yet known to be catalysed by RNA. And replication is ditions, catalytic surfaces and circumstances can be explored. The just the beginning. To successfully make the transition to the study of prebiotic provides key insights into the plaus- protein–nucleic-acid world, RNA must have been able to promote ibility of a given scenario, but it cannot hope to recreate a scenario coded peptide synthesis and a of metabolic reactions. de novo. Without constraints from phylogeny and palaeontology, and without a guide through the maze of prebiotic possibilities, The ÔRNA worldÕ theme park theorists formulating scenarios for the early evolution of life have Can RNA catalyse the reactions needed for self-replication on the started with the basic principles of replication and evolution. early Earth? Can RNA-based life achieve the metabolic sophisti- cation needed to give birth to the protein–nucleic-acid world? In The RNA world beginning to answer these questions, it has been useful to look One hypothesis is that early life was based on RNA4. That is, beyond the ribozymes found in contemporary biology – primarily was performed by catalytic rather than by protein because only seven different types have been found. Two types . The appeal of RNA-based life is that catalytic RNAs, perform self-splicing reactions, four perform self-cleavage and which could have served as their own genes, would have been one trims off the 5Ј end of pre-tRNA12. All perform phospho- much simpler to duplicate than . According to this theory, diester transfer or phosphodiester at RNA and, some- RNA first promoted the reactions required for life with the help of , DNA linkages, although valiant efforts have extended metals, pyridines, amino acids and other small- cofactors. reactions catalysed by derivatives of these ribozymes to include Then, as became more complex, RNA developed reactions at carbonyl centres13,14. Although the reactions David P. Bartel the ability to synthesize coded polypeptides that served as more of natural ribozymes are fascinating and impressive, they do not [email protected] sophisticated cofactors. DNA eventually replaced RNA as the approach the sophistication of the key reactions assumed by the Peter J. Unrau genetic polymer, and protein replaced RNA as the prominent ‘RNA world’ hypothesis. [email protected]. biocatalyst. The conversion to protein is not considered The ability to explore the repertoire of RNA catalysis dramati- edu complete; RNA retains a central role in protein synthesis, perhaps cally improved with the development of in vitro randomization, Whitehead Institute including catalysis of peptidyl transfer5. Remnants of ancestral ribo- selection and amplification methods15–17. Ribozymes with new or for Biomedical zymes are also thought to persist as within many co- enhanced activities can be isolated from large libraries of Research and Dept ϩ 18–20 factors, such as NAD , NADPH, FAD, , coenzyme B12, variants , and entirely new ribozymes can be isolated from of Biology, MIT, 6 21 ATP and S-adenosylmethionine . large pools of random-sequence molecules (Table 1). During 9 Cambridge 7 Now that the ‘RNA world’ hypothesis has been canonized in vitro selection of ribozymes, the desired sequences are enriched Center, Cambridge, within most current biology textbooks, its status as a hypothesis is on the basis of their ability to modify themselves in such a way MA 02142, USA.

TCB, Vol. 9, N¼ 12 (0962-8924) TIBS, Vol. 24, N¼ 12 (0968-0004) TIG, Vol. 15, N¼ 12 (0168-9525) © 1999 Elsevier Ltd. All rights reserved. For article fee, see p. IV. PII: S0962-8924(99)01669-4 PII: S0968-0004(99)01483-8 PII: S0168-9525(99)01898-3 M9 Millennium issue David P. Bartel and Peter J. Unrau ¥ Early RNA-based life?

‘RNA world’ hypothesis cannot be proven, recreating key features TABLE 1. Examples of new ribozymes from random-sequence a of an RNA world would make the hypothesis much more credible, RNA selections and such efforts do enforce a more rigorous focus on the relevant Bond formedb Leaving group Activity of ribozyme Refs issues and a deeper appreciation of the complexities of even the simplest imaginable life. An assembly of molecules with life-like -OÑPO -5Ј-RNA Phosphodiester cleavagec 18,48,49 3 properties, although a distant prospect, would be a fascinating HOÑPO - Cyclic phosphate hydrolysisc 18 3 tool for studying the basic properties and processes of life. Other -OÑPO3-PPi RNA ligation 21,28,44 -OÑPO3-PPi Limited polymerization 29 experimentalists are primarily interested in the fundamental -OÑPO - AMP RNA ligation 50 3 properties and possibilities of RNA, and how they compare with -OÑPO - ADP RNA phosphorylation 37 3 those of other , such as protein. They want to place -OÑPO3- Imidizole Tetraphosphate cap formation 51 d -OÑPO3- Rpp Phosphate anhydride transfer and hydrolysis 40 current biocatalysts in the context of what is possible and would Ͻ c -OÑPO3 PPi RNA branch formation 52 be pursuing new ribozymes even if there was no ‘RNA world’ -OÑCO- AMP RNA aminoacylation 36 hypothesis. Others are interested in building the technology for -OÑCO- 3Ј-RNA Acyl transfer 32 generating enzymes with new or enhanced reaction chemistries, -OÑCO- AMP Acyl transfer 38 preferences or reaction conditions. A selected in vitro has already become a useful research tool27, and -HNÑCO- 3Ј-RNA Amide bond formation 32 -HNÑCO- AMP formation 26 enzymes selected in vitro might eventually find uses as diagnostic, therapeutic or synthesis tools. Ͼ NÑCH2- I RNA alkylation 53 With this combination of motives, important elements of the -SÑCH - Br Thio alkylation 54 2 putative RNA world are under construction. At best, these cumu- ϾHCÑCHϽ DielsÑAlder addition (anthraceneÑmaleimide) 25 lative efforts will resemble an ‘RNA world’ theme park – artificial and fragmented when compared with the real thing, but still well ϾNÑCHϽ PP Glycosidic bond formation 24 i worth a visit. The second part of this article highlights the main Bridged biphenyl isomerization 42 attractions under construction: replication, coded peptide syn- thesis and several metabolic activities presumed by the ‘RNA Porphyrin metalation 43 world’ scenario. aActivities have also been isolated from pools of DNA (Ref. 55) and other RNA analogues22,23. bThe attacking nucleophile is on the left. The bond formed is indicated by an en-rule (Ñ). RNA self-replication Neighbouring bonds to one (-) or two (Ͻ or Ͼ) adjacent are indicated. The ‘RNA world’ hypothesis hinges on the assumption that some- cReactions promoted by new ribozyme folds isolated from libraries biased towards natural folds. dR can be any of a large number of moieties. where, among all RNA-sequence possibilities, ribozymes exist that can replicate RNA. The fact that some RNAs promote the chem- istry of polymerization is illustrated by a ribozyme that ligates Ͼ Ϫ1 that they can be separated from the inactive molecules. When RNA efficiently (kcat 1 sec ) using a reaction similar to the examining reactions that do not involve substrates, addition of a single during RNA polymerization28. the desired substrate can be linked to each of the random- Because the ends of the RNA molecules joined are aligned sequence-pool molecules, and molecules that convert the tethered by Watson–Crick pairing, it is possible to engineer this ribozyme substrate to product are selected22–26. Next, the selected molecules to perform limited RNA polymerization29. In the presence of are amplified, and the selection–amplification procedure is triphosphates and the appropriate template RNA, the repeated until sequences with the desired activity dominate the ribozyme extends an RNA primer by successive addition of three pool. Before the technology of in vitro selection existed, it was mononucleotides. easy to proclaim boldly that RNA could catalyse the reactions This polymerase activity is still far from that needed for self- required in the RNA world – no one expected experimental verifi- replication. It is slow, with a three-nucleotide extension some- cation. However, now the onus is not merely to propose a key times requiring more than 24 hours. Its error rate during polym- reaction of the RNA world but also to produce an RNA molecule erization is at least five times higher than that required for that can perform such a reaction. self-replication of an RNA of its size30. Most problematic, it rec- New ribozymes bring important insights into the feasibility of ognizes a specific unpaired segment of the template RNA, which the ‘RNA world’ hypothesis, but the in vitro selection approach restricts translocation and reduces the number of compatible has limitations. Even if it can demonstrate that there are RNA template sequences. The fact that other RNA enzymes can make sequences that can catalyse the reaction in question, the possi- generic contacts when recognizing substrate RNAs has been bility that other ribozymes with different folds could also promote demonstrated with derivatives of self-splicing introns31. the reaction precludes any claims that a particular fold would In summary, three key features of an RNA replicase currently have been relevant in early evolution. Moreover, it cannot prove reside in three different ribozymes and reactions. One efficiently that the RNA world ever existed. Even if ribozymes for all the catalyses the proper chemistry, another uses nucleoside triphos- essential activities of an RNA world were generated and assembled phates in a templated fashion, and the third recognizes an RNA into RNA-based life, this would only show that the fundamental duplex without regard for sequence. To prove the replicase properties of RNA are compatible with the ‘RNA world’ scenario. assumption of the ‘RNA world’ hypothesis, these features must be Perhaps most disconcerting is that the in vitro selection approach united into a single ribozyme, template fidelity must be improved cannot disprove the ‘RNA world’ hypothesis. Only a minute frac- and the issue of strand displacement must be addressed30. tion of the possible RNA sequences can be sampled in each experiment and, therefore, a negative result does not mean that RNA-catalysed protein synthesis the activity is absent among all possible sequences. Just as the replicase ribozyme would have marked the beginning Despite these limitations, an increasing number of research of an RNA world, the of coded protein synthesis groups are isolating new ribozymes. For some, the issue of the would herald the beginning of the protein–nucleic-acid world. origin and early evolution of life is such an important question The fact that RNA can catalyse the chemistry of is that it must be approached by all possible avenues. Even if the demonstrated by ribozymes that promote the formation of amide

M10 David P. Bartel and Peter J. Unrau ¥ Early RNA-based life? Millennium issue

NTPs NTPs

NTPs Rz2

Rz2 NTPs Pol Pol PPi NTPs Pol Rz2 cRz2 PPi PPi Pol cPol Pol NTPs PPi cRz2 Pol NTPs cPol

NTPs

NTPs NTPs NTPs

TCB•TIBS•TIG FIGURE 1. Example of a minimal ribo- that might eventually be constructed in the lab, provided that suitable ribozymes and membrane component can be identified. Although clearly not an , and likely to be different from the first life of the RNA world, such an organism would display the basic features of life. A polymerase ribozyme (Pol) uses nucleoside triphosphates (NTPs) to make more copies of itself and its complement (cPol) in an autocatalytic cycle (purple arrows). The polymerase ribozyme also synthesizes ribozyme 2 and its complement (Rz2 and cRz2, respectively; green arrows). Ribozyme 2 promotes a reaction that facilitates growth and eventual division of the vesicle. In this example, ribozyme 2 converts a precursor molecule (square heads) to the membrane component (round heads; red arrows). The vesicle membrane is permeable to nucleoside triphosphates and membrane precursor but impermeable to . linkages22,26,32. For example, a ribozyme has been selected that ribozymes are presumed to have promoted oxidation and reduc- forms a peptide linkage between a moiety attached to tion reactions, aldol and Claisen condensations, transmethylations the ribozyme and any one of several biotinylated amino acids26,33. and porphyrin biosynthesis35. At least, ribozymes would have had The biotinylated was activated by adenosine in a to synthesize any nucleotides, , amino acids and cofactors manner analogous to activation of amino acids by tRNA. that could not be scavenged from the environment. Ribozymes It would be interesting to explore whether this ribozyme can be would also have had to activate nucleotide and amino acid a starting point for generating ribozymes capable of coded oligo- for polymerization, presumably by taking advantage peptide synthesis. Unlike replicase activity, modern protein syn- of metabolism based on nucleotide phosphates35. thesis might still harbour remnants of RNA catalysis, suggesting Ribozymes promoting several of these classes of chemical trans- another approach for generating coded peptide synthesis: it might formations have been isolated from random sequences. Ribozymes be possible to start with ribosomal RNA sequences and work back have been found that aminoacylate themselves36. Aminoacylation towards an RNA-only activity. It is encouraging that, when the RNA of tRNA is the second step of the reaction needed to activate amino of the ribosomal large subunit is stripped of all but a few of its pro- acids for translation. Other RNAs promote the chemistry required teins, it still promotes a non-coded peptidyl transfer reaction, which for glycosidic bond formation, a key reaction in nucleotide syn- is considered a model for the chemistry of protein synthesis34. thesis24. Hydroxyl phosphorylation37 and acyl transfer32,38 reactions are also among the catalytic repertoire of RNA. RNA-based metabolism Important classes of reactions yet to be demonstrated include Significant metabolic complexity would have been required for oxidation–reduction reactions and reactions involving – RNA-based life to develop coded protein synthesis sufficient for carbon bonds (beyond Diels–Alder reactions23,25). Particularly the transition to protein-based metabolism. Although these meta- crucial are those reactions that illustrate how ribo- bolic reactions do not present the coding and translocation intri- could have captured energy from chemicals in their environment cacies of RNA polymerization and translation, they offer a unique and used it to generate a compound such as ATP, suitable for set of challenges, typically involving more difficult chemical trans- facilitating otherwise disfavoured reactions. To meet life’s meta- formations as well as recognition of small-molecule substrates. bolic challenges, protein enzymes pick from an assortment of The argument that the nucleotides of cofactors are metal and organic cofactors. RNA enzymes normally use metal remnants of the RNA world6 implies that key reactions involving cofactors but not organic cofactors12. The use of organic cofactors these cofactors were once catalysed by RNA35. Specifically, was illustrated recently by a DNA enzyme that requires histidine

M11 Millennium issue David P. Bartel and Peter J. Unrau ¥ Early RNA-based life?

as a during RNA cleavage39. A further step would be to Minimal RNA-based life show that the use of organic cofactors can extend the scope of Impressive as the current ‘RNA world’ theme park might be, all RNA catalysis beyond that seen with metal cofactors. of its displays must be considered ‘under construction’. There is Although some key reactions of the ‘RNA world’ hypothesis, still no example of a ribozyme with sufficient significance and most notably RNA polymerization, aminoacylation and trans- efficiency that it could functionally substitute for an activity pre- lation, involve RNA or derivatized RNA, typical metabolic sumed by the ‘RNA world’ hypothesis. The generation of more- reactions involve small molecules that are not attached to RNA. relevant activities will enable integration of the separate displays Demonstrating that RNA can promote an interesting reaction within the theme park into more sophisticated and convincing using a tethered substrate is important but is only the first step in attractions, such as RNA-based metabolic pathways. addressing the question of whether RNA can catalyse a reaction A particularly intriguing combination of two ribozyme activ- involving relevant small molecules. For example, some RNA ities is inspired by properties of vesicles made of long-chain fatty sequences can phosphorylate an RNA oligonucleotide37, raising acids: one ribozyme would synthesize the vesicle membrane com- the question of whether any RNAs could phosphorylate free ponent, and a second ribozyme would replicate itself and the first . Addressing this is challenging because the most produc- ribozyme (Fig. 1). Vesicles made of oleic acid grow and divide in tive approach for isolating ribozymes requires that one of the sub- the presence of a precursor that hydrolyses to form additional strates be attached to RNA. In a few cases, the molecule originally fatty acid46. These vesicles retain RNA yet are attached to the ribozyme can be removed and used as a substrate somewhat permeable to ATP46, and thus it is possible to imagine when added free in solution29,33,40. However, conversion of other a vesicle that could enclose the two ribozymes without ribozymes to forms that use free small-molecule substrates might excluding access to NTPs and membrane precursor supplied as require the development of selection protocols that involve enclosure nutrients in the medium. If fed these nutrients, the vesicles with within membranes in order to discriminate between ribozyme ribozymes that synthesize membrane component would grow variants based on colocalization with their small-molecule prod- and divide. Long-term growth and would depend ucts41. Another approach might be to isolate RNAs based on their on the activities of both ribozymes, and vesicles with improved ability to bind transition-state analogues42,43, but this strategy has ribozymes would enjoy a reproductive advantage. Such improved not yet generated ribozymes able to form new covalent bonds. ribozymes might emerge from the variation generated from RNA As RNA-based metabolic pathways bifurcated and formed net- replication errors. RNAs with entirely new activities might also works, regulation would have become increasingly important in the emerge: for example, RNAs that selectively increase membrane RNA world. In this regard, it is interesting that allosteric ribo- permeability47 to NTPs. With all the elements of darwinian evo- zymes have been generated with activities modulated dramatically lution in place, those scientists constructing the ‘RNA world’ by the presence of organic small-molecule effectors, such as adenosine might be able to take a break (except to feed the vesicles NTPs or theophylline44,45. This evokes a scenario with ribo-organisms able and membrane precursor) and watch as their RNA world assumes to respond rapidly to environmental changes or internal needs. some of the responsibility for its own construction.

References 14 Joyce, G.F. et al. (1996) Cleavage of an 27 Santoro, S.W. and Joyce, G.F. (1997) A 1 Deckert, G. et al. (1998) The complete amide bond by a ribozyme: correction. general purpose RNA-cleaving DNA enzyme. of the hyperthermophilic bacterium Science 272, 18Ñ19 Proc. Natl. Acad. Sci. U. S. A. 94, Aquifex aeolicus. 392, 353Ñ358 15 Tuerk, C. and Gold, L. (1990) Systematic 4262Ñ4266 2 Schopf, J.W. (1993) Microfossils of the early evolution of ligands by exponential enrichment 28 Ekland, E.H. et al. (1995) Structurally Archean apex chert Ñ new evidence of the Ñ RNA ligands to bacteriophage-T4 complex and highly-active RNA antiquity of life. Science 260, 640Ñ646 DNA-polymerase. Science 249, 505Ñ510 derived from random RNA sequences. Science 3 Mojzsis, S.J. et al. (1996) Evidence for life on 16 Robertson, D.L. and Joyce, G.F. (1990) 269, 364Ñ370 Earth before 3,800 million years ago. Nature Selection in vitro of an RNA enzyme that 29 Ekland, E.H. and Bartel, D.P. (1996) 384, 55Ñ59 specifically cleaves single-stranded DNA. RNA-catalysed RNA polymerization using 4 Joyce, G.F. and Orgel, L.E. (1999) in The RNA Nature 344, 467Ñ468 nucleoside triphosphates. Nature 382, World (Gesteland, R.F., Cech, T.R. and 17 Ellington, A.D. and Szostak, J.W. (1990) 373Ñ376 Atkins, J.F., eds), pp. 49Ñ77, Cold Spring In-vitro selection of RNA molecules that bind 30 Bartel, D.P. (1999) in The RNA World Harbor Press specific ligands. Nature 346, 818Ñ822 (Gesteland, R.F., Cech, T.R. and Atkins, J.F., 5 Green, R. and Noller, H.F. (1997) 18 Pan, T. and Uhlenbeck, O.C. (1992) A small eds), pp. 143Ñ162, Cold Spring Harbor and translation. Annu. Rev. Biochem. 66, metalloribozyme with a 2-step mechanism. Laboratory Press 679Ñ716 Nature 358, 560Ñ563 31 Doudna, J.A. and Szostak, J.W. (1989) 6 White, H.B. (1976) Coenzymes as fossils of an 19 Beaudry, A.A. and Joyce, G.F. (1992) Directed RNA-catalysed synthesis of earlier metabolic state. J. Mol. Evol. 7, evolution of an RNA enzyme. Science 257, complementary-strand RNA. Nature 339, Acknowledgements 101Ñ104 635Ñ641 519Ñ522 We thank 7 Gilbert, W. (1986) Origin of life Ñ the RNA 20 Lehman, N. and Joyce, G.F. (1993) Evolution 32 Lohse, P.A. and Szostak, J.W. (1996) M. Lawrence, world. Nature 319, 618 in vitro of an RNA enzyme with altered metal Ribozyme-catalysed amino-acid transfer dependence. Nature 361, 182Ñ185 reactions. Nature 381, 442Ñ444 E. Schultes, 8 Orgel, L.E. and Lohrmann, R. (1974) Prebiotic chemistry and nucleic acid replication. 21 Bartel, D.P. and Szostak, J.W. (1993) Isolation 33 Zhang, B.L. and Cech, T.R. (1998) S. Baskerville and Prebiotic Chem. 7, 368Ñ377 of new ribozymes from a large pool of random Peptidyl- ribozymes: trans other members of 9 Shapiro, R. (1988) Prebiotic ribose synthesis Ñ sequences. Science 261, 1411Ñ1418 reactions, structural characterization and the lab for helpful a critical analysis. Orig. Life Evol. 22 Wiegand, T.W. et al. (1997) Selection of RNA ribosomal RNA-like features. Chem. Biol. 5, amide syntheses. Chem. Biol. 4, 675Ñ683 539Ñ553 comments on this 18, 71Ñ85 10 Levy, M. and Miller, S.L. (1998) The stability 23 Tarasow, T.M. et al. (1997) RNA-catalysed 34 Khaitovich, P. et al. (1999) Characterization manuscript. This of the RNA bases: Implications for the origin carbonÑcarbon bond formation. Nature 389, of functionally active subribosomal work was supported of life. Proc. Natl. Acad. Sci. U. S. A. 95, 54Ñ57 from Thermus aquaticus. Proc. Natl. Acad. by grants from the 7933Ñ7938 24 Unrau, P.J. and Bartel, D.P. (1998) Sci. U. S. A. 96, 85Ñ90 RNA-catalysed nucleotide synthesis. Nature 35 Benner, S.A. et al. (1993) in The RNA World NIH and NASA to 11 Eschenmoser, A. (1999) Chemical etiology of nucleic acid . Science 284, 395, 260Ñ263 (Gesteland, R.F. and Atkins, J.F., eds), D.P.B. and a 2118Ñ2124 25 Seelig, B. and Jaschke, A. (1999) A small pp. 27Ñ70, Cold Spring Harbor Laboratory Press postdoctoral 12 Pyle, A.M. (1993) Ribozymes Ñ a distinct class catalytic RNA motif with Diels-Alderase 36 Illangasekare, M. et al. (1995) Aminoacyl-RNA fellowship from the of metalloenzymes. Science 261, 709Ñ714 activity. Chem. Biol. 6, 167Ñ176 synthesis catalysed by an RNA. Science 267, 26 Zhang, B.L. and Cech, T.R. (1997) Peptide 643Ñ647 Medical Research 13 Piccirilli, J.A. et al. (1992) Aminoacyl esterase-activity of the bond formation by in vitro selected 37 Lorsch, J.R. and Szostak, J.W. (1994) In-vitro Council (Canada) to ribozyme. Science 256, 1420Ñ1424 ribozymes. Nature 390, 96Ñ100 evolution of new ribozymes with P.J.U.

M12 David P. Bartel and Peter J. Unrau ¥ Early RNA-based life? Millennium issue

kinase activity. Nature 371, 31Ñ36 44 Robertson, M.P. and Ellington, A.D. (1999) optimum. Proc. Natl. Acad. Sci. U. S. A. 94, 38 Jenne, A. and Famulok, M. (1998) A novel In vitro selection of an allosteric ribozyme 10612Ñ10617 ribozyme with ester transferase activity. that transduces analytes to amplicons. Nat. 50 Hager, A.J. and Szostak, J.W. (1997) Isolation Chem. Biol. 5, 23Ñ34 Biotechnol. 17, 62Ñ66 of novel ribozymes that ligate AMP-activated 39 Roth, A. and Breaker, R.R. (1998) An amino 45 Soukup, G.A. and Breaker, R.R. (1999) RNA substrates. Chem. Biol. 4, 607Ñ617 acid as a cofactor for a catalytic Engineering precision RNA molecular 51 Chapman, K.B. and Szostak, J.W. (1995) polynucleotide. Proc. Natl. Acad. Sci. U. S. A. switches. Proc. Natl. Acad. Sci. U. S. A. 96, Isolation of a ribozyme with 5ЈÑ5Ј 95, 6027Ñ6031 3584Ñ3589 activity. Chem. Biol. 2, 325Ñ333 40 Huang, F.Q. et al. (1998) RNA enzymes with 46 Walde, P. et al. (1994) OparinÕs reactions 52 Tuschl, T. et al. (1998) Selection in vitro of two small-molecule substrates. Chem. Biol. 5, revisited: Enzymatic synthesis of novel ribozymes from a partially randomized 669Ñ678 poly(adenylic acid) in micelles and self- U2 and U6 snRNA library. EMBO J. 17, 41 Tawfik, D.S. and Griffiths, A.D. (1998) Man- reproducing vesicles. J. Am. Chem. Soc. 116, 2637Ñ2650 made cell-like compartments for molecular 7541Ñ7547 53 Wilson, C. and Szostak, J.W. (1995) In-vitro evolution. Nat. Biotechnol. 16, 652Ñ656 47 Khvorova, A. et al. RNAs that bind and change evolution of a self-alkylating ribozyme. 42 Prudent, J.R. et al. (1994) Expanding the the permeability of membranes. Nature 374, 777Ñ782 scope of RNA catalysis. Science 264, Proc. Natl. Acad. Sci. U. S. A. 96, 10649Ñ10654 54 Wecker, M. et al. (1996) In vitro selection of 1924Ñ1927 48 Williams, K.P. et al. (1995) Selection of novel a novel catalytic RNA: Characterization of a 43 Conn, M.M. et al. (1996) Porphyrin Mg2ϩ-dependent self-cleaving ribozymes. sulfur alkylation reaction and interaction with metallation catalysed by a small RNA EMBO J. 14, 4551Ñ4557 a small peptide. RNA 2, 982Ñ994 molecule. J. Am. Chem. Soc. 118, 49 Jayasena, V.K. and Gold, L. (1997) In vitro 55 Breaker, R.R. (1997) DNA and DNA 7012Ñ7013 selection of self-cleaving RNAs with a low pH enzymes. Curr. Opin. Chem. Biol. 1, 26Ñ31

Human evolution Svante PŠŠbo

The origin, history, and singularity of our has fascinated storytellers, philosophers and scientists throughout, and doubtless before, recorded history. Anthropology, the modern-era discipline that deals with these issues, is a notoriously contentious , perhaps because the topic at hand Ñ the nature of our own species Ñ is one that is difficult or impossible to approach in an unbiased way. Recently, molecular has increasingly contributed to this field. Here, I briefly discuss three areas where I believe molecular studies are likely to be of decisive importance in the future. These concern the questions of where and when our species originated, what the genetic background for characters that differ between us and apes is, and how the phenotypic traits that vary among groups have evolved.

tudies of the genetic variation of , the concern of the species is not trivial. However, from a molecular-genetic perspec- field of molecular anthropology, attempt to produce objective tive, it is clear that the DNA sequences found in contemporary S data with which to arrive at new insights about human history. have been passed down to them from previous gener- These insights can be of great practical importance, as in the ations. It is also clear that, in every generation, some DNA quest for genetic variation associated with disease susceptibility. sequences are not passed on because some individuals have no They can also bear on questions about human history within the children or the sequence fails to be transmitted during . past few thousand years, such as the colonization of previously Therefore, the genealogy of a DNA sequence will trace back to fewer uninhabited areas and subsequent migrations. However, for many and fewer ancestors until it comes together in one common ancestor. of these issues, other sources of knowledge, such as archaeological To reconstruct this genealogy, the most straightforward approach or historical records, can often be of equal or greater importance. is to determine DNA sequences from individuals that are distrib- Here, rather, I would like to discuss briefly three questions for uted such that they represent the entire species. We can then use which molecular studies are likely to be of decisive importance. mathematical techniques to estimate the age of the most recent Where do we come from? Why do we look different from one common ancestor of this collection of contemporary DNA se- another? Why are we so different from other species? First, I will quences. However, because the genealogy of sequences at different outline how we have begun recently to understand when and locations in the genome differs owing to recombination and segre- where the earliest genetic differences that occur in our pool gation, the age and place of the origin will be different for each emerged. Then, I describe the early beginnings of insights into genetic locus. Thus, from a genetic perspective, there will not be a the genetic background of one of the most obvious differences single answer to the question of when and where our species among humans – that of pigmentation – and, finally, I discuss emerged. Only if many loci show the same or a similar pattern can Svante PŠŠbo how we might hope to approach the as-yet-unknown genetic one infer that some kind of a phenomenon occurred, [email protected] foundations of the differences between our own species and our as such an event would affect several parts of the genome. Max Planck Institute closest evolutionary relatives, the African apes. The mitochondrial genome is the locus for which the most for Evolutionary on DNA sequence diversity in humans is currently Anthropology, Origins of human genetic variation available. The great majority of estimates of the age of the deepest Inselstrasse 22, The questions of when and where our species originated might seem divergence among human mitochondrial genomes fall between D-04103 Leipzig, quite straightforward, but, in fact, the definition of the origin of a 100 000 and 200 000 years ago (for a review, see Ref. 1). When a Germany.

TCB, Vol. 9, N¼ 12 (0962-8924) TIBS, Vol. 24, N¼ 12 (0968-0004) TIG, Vol. 15, N¼ 12 (0168-9525) © 1999 Elsevier Science Ltd. All rights reserved. For article fee, see p. IV. PII: S0962-8924(99)01688-8 PII: S0968-0004(99)01497-8 PII: S0168-9525(99)01904-6 M13