Over the past decade, researchers haveapplied the principles of darwinianevolution in the laboratory to generate
hundreds of RNA sequences that fold intounique shapes and thereby bind to specifictarget molecules. The experiments revealedthe capacity of these folded RNAs — com-monly known as aptamers — to recognizevirtually any molecular target, but led to a growing mystery: why is this ability notused in nature? Two papers from a group ledby Ron Breaker, published on page 952 of thisissue1 and in Chemistry and Biology 2, nowdissolve this conundrum, by showing thatregulatory sequences in certain bacterialmessenger RNAs can in fact sense small molecules directly.
Like most biological processes, the bio-synthesis and import of vitamins B1 (thia-mine and its active form, thiamine pyro-phosphate), B12 (adenosyl-cobalamin) andB2 (riboflavin) by bacteria requires specificenzymatic and transport proteins, which areproduced from genes via messenger RNA
intermediates. These mRNAs contain pro-tein-coding stretches of sequence, as well asnon-protein-coding stretches3–5. The non-coding sequences are needed to controltranslation of the mRNAs into proteins:they are part of signal-transduction path-ways that sense the level of a given metabo-lite (the relevant vitamin), and use thatinformation to control the efficiency ofenzyme production.
Normally (that is, in all examples knownuntil now), control regions such as theseare bound by metabolite-sensing proteins,which regulate the conformation of themRNA and thus the access of the ribosome— the protein-making machine — to trans-lation-initiation signatures within themRNA. Such regulatory proteins had beensought for bacterial vitamin biosynthesis,but had not been found. So it was proposedthat in these cases the RNA might interactdirectly with its cognate metabolite3–5
(reviewed in ref. 6). Support for this hypoth-esis was particularly strong in the case of vita-
min B12, which could inhibit ribosome bind-ing to a relevant mRNA in a purified systemcontaining just ribosomes and mRNA.
To obtain more direct evidence for anRNA–metabolite interaction, Winkler et al.1
and Nahvi et al.2 synthesized the previouslydefined regulatory regions of two mRNAscoding for enzymes involved in B1 bio-synthesis, and one mRNA that codes for a B12 transporter, respectively. The authorsproved that a direct interaction occurred byobserving changes in the pattern of sponta-neous, hydroxide-ion-mediated cleavageof the mRNAs in the presence of the targetvitamin. Independent confirmation ofRNA–metabolite binding was provided byequilibrium dialysis.
The patterns of spontaneous cleavageevents also helped the authors to define thesecondary structures — the patterns of base-pairing between segments of the RNA —of the active, folded RNA sequences. The vitamin-dependent changes in these pat-terns pointed to a conformational alterationin the folded state of the RNAs upon vitaminbinding. These data showed that a transla-tion-initiation signature known as theShine–Dalgarno ribosome-binding site wasaccessible in the unbound state, but becameoccluded in the bound state (Fig. 1). Winkleret al. and Nahvi et al. then generated a series ofmutant RNAs that were expected to favourone of these two states of the Shine–Dalgarno
groups and the hydrogen-bonding OHgroups are sandwiched in between. So in thiscase, the surface reconstruction need notinterrupt the linear chains of hydrogenbonds formed within the ‘sandwich’.
The observations made by Wilson et al.show directly that the intermolecular sepa-ration in water at the surface is about 5%greater than in the bulk liquid, and so is simi-lar to that of a gas-phase dimer. At the surfaceof methanol, however, the separation is about5% lower, reaching a value close to that forthe crystalline solid. In water, the increasinglyorganized hydrogen-bonded structure, fromdimer to liquid to ice, leads to increasinglystronger attractive forces and increasinglyshorter intermolecular distances. The resultsof the EXAFS study reflect the decrease inorganized hydrogen bonding at the watersurface and the increase in order at the sur-face of liquid methanol.
The broader impact of this new EXAFSexperiment will depend on how well it can be generalized to describe solutions. Amongthe implications for interfacial chemistry,the direct observation of the solvation ofsurface-adsorbed species is a potential out-come. The method measures the averagesurroundings of all atoms of the same ele-mental type, so a new focus must be on sys-tems in which the solute has a distinguished
atom, of different elemental type. Ionic solu-tions are a practical example in atmosphericchemistry, as are solutes with distinguishedatoms such as chlorine.
Although interatomic distances have adistinct signature in EXAFS, the composi-tion of the local environment cannot at present be well determined in this surfacecase. One need, and challenge, is a new theo-retical description of EXAFS signals inregions of inhomogeneous density — densi-ty that at a liquid surface varies very rapidlyindeed, from liquid to vacuum in less than ananometre. It is clear that the variousapproaches to interfacial characterizationwill continue to be complementary. A syner-gistic element that should not be underesti-mated is that the present work and furtherapplications will provide a benchmark fordirect tests of the molecular models thatunderpin atomistic simulation. ■
Peter J. Rossky is in the Department of Chemistryand Biochemistry, University of Texas, Austin, Texas 78712-1167, USA.e-mail: [email protected]
1. Wilson, K. R. et al. J. Chem. Phys. 117, 7738–7744 (2002).
2. Nathanson, G. M., Davidovits, P., Worsnop, D. & Kolb, C. E.
J. Phys. Chem. 100, 13007–13020 (1996).
3. Benjamin, I. Acc. Chem. Res. 28, 233–239 (1995).
4. Schlossman, M. Curr. Opin. Coll. Int. Sci. 7, 235–243 (2002).
5. Penfold, J. Rep. Prog. Phys. 64, 777–814 (2001).
6. Richmond, G. Annu. Rev. Phys. Chem. 52, 357–389 (2001).
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890 NATURE | VOL 419 | 31 OCTOBER 2002 | www.nature.com/nature
Molecular biology
RNA gets a grip on translationJack W. Szostak
These are interesting times for those who study RNA. The latest curiousdiscovery is that some messenger RNAs have sequences that sense smallmolecules directly, so controlling translation of the RNA into protein.
5'
5'
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Go!
Stop!
SD
SD
ThiPP
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Figure 1 RNA as metabolite sensor andtranslation regulator. The figure representspart of a messenger RNA that encodes abacterial protein involved in the biosynthesis ofvitamin B1 (thiamine pyrophosphate, ThiPP).This part contains a region involved in sensingThiPP; the so-called Shine–Dalgarno (SD)sequence, which is recognized by the ribosome;and a sequence that marks where the enzyme-encoding portion of the mRNA begins.a, Winkler et al.1 have found that in the absenceof ThiPP, the sensor region adopts a confor-mation that exposes the Shine–Dalgarnosequence. This would allow the ribosome tobind and begin translation. b, Binding to ThiPPcauses the sensor region to change shape,obscuring the Shine–Dalgarno sequence. ThusThiPP controls the production of one of itsbiosynthetic enzymes directly via a sequencewithin the enzyme-encoding mRNA.
© 2002 Nature Publishing Group
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NATURE | VOL 419 | 31 OCTOBER 2002 | www.nature.com/nature 891
Recycling campaigners andenvironmental groups often quotealarming statistics on how muchwaste one person produces eachyear. But a comprehensive report on rubbish collected in New YorkCity throughout the twentiethcentury claims that the figure hasdropped dramatically, from its peakin the 1940s.
Writing in Environmental Scienceand Technology (doi: 10.1021/es011074t), Daniel Walsh describesthe rise and fall of garbagecomponents in the Big Apple. Usingthe most complete set of municipalrecords for a US city’s residentialrefuse, Walsh records a maximumoutput of 940 kg of waste per personin 1940, and a low of 320 kg perperson in both 1961 and 1963.
Surprisingly, since the 1980s aperson’s annual throwaways havestabilized at a relatively low 430 kg.Also, the most significant trashtrends were mostly declines in thepercentages of different categoriesof waste. The relative amounts offuel ash, food waste, metal and
glass dropped, but the percentage of plastics rose and that of paperremained the same. The photographon the left shows ash collectionearlier in the twentieth century; thaton the right a present-day scene.
Between 1920 and 1990 therewas a 50% decrease in refusedensity. Walsh attributes this to adecrease in coal and other fuel ash,
and to technologies that haveenabled product packaging toswitch from glass and metal topaper and plastic. Over the sameperiod, organic-matter waste rosefourfold, increasing the greenhouse-gas potential per unit of dumped or incinerated garbage. Walshestimates that the totality of NewYork City’s refuse for the past
century represents a carbon pool of80 million tons.
As coal was at the start of thetwentieth century, paper is now themost abundant category of refuse,accounting for roughly 35% of allresidential discards. Will the figuresfor the twenty-first century reflectthe long-forecast advent of thepaperless office? Kendall Powell
Environment
Trash trends
sequence. The results strongly supported thehypothesized vitamin-controlled structuraltransition, explaining the observed control of mRNA translation. Similar evidence hasbeen claimed (but not yet published) for anmRNA control region required in vitamin B2
biosynthesis.One puzzle is that the vitamin-sensing
mRNA sequences that control translation1,2
are considerably larger, at up to 200nucleotides, than the aptamers that havebeen generated in vitro, which typically con-sist of less than 40 nucleotides. Why is this?The most likely explanation is that themRNA control regions do much more thanjust bind a target molecule. They functionby virtue of their ability to fold into twodistinct conformations, which are finelybalanced energetically so that a change invitamin concentration drives the transitionfrom one conformation, which allows trans-lation to begin, to the other, which does not.However, several groups, including Break-er’s, have ‘evolved’ RNA elements in vitrothat can switch between conformations in aligand-dependent way, yet are much smallerthan the in vivo examples. Perhaps the largesize of the in vivo regulatory sequences is an‘in vivo artefact’ of the constraints on theevolution of functional RNA structuresfrom a limited set of initial sequences. Incontrast, the in vitro situation generally
involves sampling from a large set of ran-dom sequences.
Why have metabolite-sensing RNAsequences been found (so far) only inmRNAs involved in vitamin biosynthesisand import? One intriguing possibility, sug-gested by Winkler et al.1, is that these RNAcontrol elements are ancient, dating from the‘RNA world’ — a hypothesized early stage inthe evolution of life on Earth, when proteinsdid not exist. All three vitamins in questionare biochemically more or less RNA-like (B12
contains an adenosine group), and havethemselves been proposed to date from theRNA world7. But many other metabolites,and their biosynthetic pathways, must be asold or older. With so much of biochemistrynow subject to the domination of proteins, itis unclear why the biosynthesis of these par-ticular metabolites should have remained sostaunchly within the realm of RNA. Perhapsthese issues will be clarified if and when addi-tional examples of RNA-mediated metaboliccontrol are discovered.
Whatever the answer, it is clear thatresearchers investigating translational con-trol must bear in mind that non-codingmRNA sequences might regulate thisprocess directly. A different example of thisphenomenon was provided recently byJohansson et al.8, who described an mRNAregulatory sequence that acts as a direct tem-
perature sensor in a pathogenic bacterium,Listeria, controlling translation withoutneeding any regulatory proteins. This RNAsensor detects the increase in temperaturethat occurs when the bacterium moves into amammalian host, and regulates the expres-sion of genes associated with bacterial viru-lence. Along with the discovery of thenumerous, widespread micro-RNA molec-ules, which seem to regulate the translationof mRNAs in a variety of ways, these findingsprovide striking examples of new and unex-pected roles for RNA in controlling geneexpression. Given the current pace of discov-ery, it seems likely that yet more surprisesmay be just around the corner. ■
Jack W. Szostak is at the Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.e-mail: [email protected]. Winkler, W., Nahvi, A. & Breaker, R. R. Nature 419,
952–956 (2002); advance online publication, 16 October
2002 (doi:10.1038/nature01145).
2. Nahvi, A. et al. Chem. Biol. 9, 1043–1049 (2002).
3. Nou, X. & Kadner, R. J. Proc. Natl Acad. Sci. USA 97,
9170–9175 (2000).
4. Miranda-Rios, J., Navarro, M. & Soberón, M. Proc. Natl
Acad. Sci. USA 98, 9736–9741 (2001).
5. Gelfand, M. S. et al. Trends Genet. 15, 439–442 (1999).
6. Stormo, G. D. & Yongmei, J. Proc. Natl Acad. Sci. USA 98,
9465–9467 (2001).
7. White, H. B. III J. Mol. Evol. 7, 101–104
(1976).
8. Johansson, J. et al. Cell 110, 551–561 (2002).
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