TIBS 14 - December 1989 505

Reflections on Biochemistry Creation of a general model of

RNA spatial organization AJLexey A. Bogdanov

It was exactly a hundred years ago that Richard Altmann, Professor of Histol- ogy at the Leipzig University, isolated a protein-free nucleic acid f~:om yeast cells 1. This preparation was later dem- onstrated to be pure ribonucleic acid, but it was about 50 years before it became clear that RNA participates somehow in protein biosynthesis 2. The key role of RNA in translation was recognized only in the late 1950s. At about the same time [due tc, efforts of the laboratories of Paul Doty (Fig. 1) at Harvard, and Alexander Spin,in (Fig. 2) at A.N. Bach Institute of Biochem- istry, Moscow] the regular macromol- ecular structure of RNA was resolved. The results of their work were summar- ized for the first time in the form of a general model of the secondary and tertiary structure of RNA in 1960 in Doty's Harvey Lecture enti',:led Inside Nucleic Acids 3, and in Spirin's review article On Macromolecular Structure o f Native High-Polymer Ribonucleic Acid in Solution 4.

Although the pioneer work by Doty's and Spirin's labs led to eluci- dation of the general principles of spatial organization of the third (after proteins and DNA) major class of bio- polymers, it is rarely mentioned in text- books and historical treatises as an important step in the development of molecular biology. No place for it was found in the symbolic Mo,!ecular bi- ology's hall of fame which was 'built' by Jan Witkowski on the occasion of the 50th anniversary of this branch of bi- ology 5.

Now that several astonishing dis- coveries have changed our view of 'The RNA World '6, it seems timely to recall how the first model of RNA macro- molecular structure was created. The model that is fundamental for studies around the world.

A. A. Bogdanov is at the A. N. Bel, gzersky Lab- oratory o f Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow 119899, USSR.

Prehistory The most important requisites for the

beginning of RNA secondary and ter- tiary structure investigations were: (1) the development of the phenol pro- cedure of nucleic acid deproteinization7; and (2) the discovery made by Gierer and Schramm s and Fraenkel-Conrat et al. 9 that RNA of tobacco mosaic virus (TMV) was infectious. Phenol deproteinization, which provides im- mediate inactivation of nucleases, made homogeneous high molecular weight RNA preparations available for physical-chemical study. The dis- covery of infectivity of the viral RNA demonstrated that the isolation pro- cedures did not impair RNA biological activity.

It was shown in the first experiments performed with intact TMV RNA that, in contrast to DNA, the ribonucleic acid is a single-stranded molecule l°.

This conclusion has been found to be a general rule for viral and cellular RNA and only several years later were some rare exceptions to this rule dis- covered 1~. On the other hand, it was shown that homopolyribonucleotides - poly(A) and poly(U) - were able to form ideal double-helical complexes ~2. Thus, the RNA chain appeared to be able, in principle, to give DNA-like structures with Watson-Crick base pairs.

Secondary structure In 1957-1958 Hall and Doty

measured the UV absorbence of RNA solutions as a function of temper- ature 13. The cooperative melting of double-helical DNA had been dis- covered in Doty's lab shortly before ~4 and they wondered if single-stranded RNA would undergo any sort of struc- tural transition upon heating. To their surprise the absorbance of ribosomal RNA appeared to increase rather sharp- ly, and RNA exhibited a characteristic melting curve 13. These findings re- ceived further support immediately: both the Harvard ~5 and MOSCOW 16A7

groups observed similar melting of TMV RNA structure. It was noted that a temperature-induced increase of

Fig. 1. Paul Doty (left) with B. Gottikh and Ya Varshavsky (right) during the International Congress of Biochemistry, Moscow, 1961.

© 1989, Elsevier Science Publishers Ltd, (UK) 0376-5067/89/$112.lX~

506 T I B S 1 4 - December 1989

Fig. 2. Alexander Spirin and his model in Moscow in the early 1960s.

RNA absorbance was accompanied by a decrease of optical activity and an increase in viscosity of their solutions. The magnitude of all these effects was shown to be dependent on ionic strength of buffers and RNA nucleo- tide composition. It was stressed, how- ever, that RNA underwent structural transitions in a much broader temper- ature range than double-helical DNA.

The similarities and differences in DNA and RNA behavior suggested that RNA molecules had to have regu- lar DNA-like regions, but that only part (estimated as 40-60%) of their chain was organized in this manner, and double-helical regions had to be different in base pair number and com- position. Since RNAs are single- stranded molecules, their DNA-like regions were thought to be formed as result of complementary A - U and G - C pairing of adjacent parts of polynucleo- tide chain. Moreover, Doty and co- workers assumed that RNA double- helical regions would not be perfect

,~,~,~ Fig. 3. The first model ~---c, o f R N A secondary

¢'u---] struclurel'~. ~?,~ ,..~

"K+" ,~ , ,v, . ~ . . . c ( ~

,~ ~,,~ ~, i---~,

helices but rather, had looped out resi- dues and other defects ~8.

The first model of the secondary structure of RNA with a random sequence of 90 nucleotides was pub- lished by Fresco, Alberts and Doty in 1960 ~s. It is shown on Fig. 3. It consists of three hairpin loops with several mis- matched nucleotide residues connected with short single-stranded regions. The model satisfied perfectly all the exper- imental data accumulated up to that time.

Tertiary structure Some of these data indicated unam-

biguously that, under physiological conditions, RNA exists in the con- figuration of a rather compact coil 14-17. With increasing temperature or decreasing counter-ion concentration the coil extended in overall size and could be converted finally into a com- pletely unfolded thread 19'2°. Fur- thermore, it was shown by Spirin and co-workers that the alterations in RNA tertiary structures started before the melting of double-helical regions and led to the appearance of some inter- mediate (probably ordered) states 4. Interestingly, the Moscow group, in collaboration with N. Kisselev, was able to follow these transitions by means of electron microscopy 2~. Such behavior of RNA tertiary structure cor- related perfectly with the proposed sec- ondary structure model. The existence of single-stranded regions, postulated

with the model, indeed allows the fold- ing of double helices into a compact coil at high ionic strength.

Continuity of RNA polynucleopeptide chain

The Harvard and Moscow groups were at variance over only one prob- lem. Doty and co-workers, as well as some other groups, had found that the molecular weight of ribosomal RNA decreased drastically after heating. Hence they proposed that RNA macro- molecules were organized from rela- tively small subunits held together with intramolecular hydrogen bonds 12. Spirin and co-workers, however, contended that such 'subunits' were artifacts which appeared because of hidden breaks produced with nu- cleases, and any RNAs isolated with all necessary precautions were covalently continuous chains 23. By 1962 Doty et al. re-examined this problem and con- cluded that RNA, carefully purified from nuclease contaminations, did not dissociate under denaturating condi- tions 24. Once again the interesting exceptions to this general rule have been found only many years later 252~'.

By the early 1960s, then, the general model which described RNA second- ary and tertiary structure had been created. The authors emphasized that the model was universal and could be applied to any type of single-stranded RNA. It was also suggested that RNAs were organized in the same way in cellular ribonucleoprotein particles, for instance, in ribosomes [although at least one example of RNA structure rearrangement in the course of inter- action with proteins (i.e. TMV recon- stitution) has been known27]. All the subsequent studies of RNA secondary and tertiary structure have proved the validity and universality of the original model in its major features.

Confirmation of the model Whereas the model described the

RNA tertiary structure in terms of overall size and shape, which could be easily measured, many features of RNA secondary structure were hypo- thetical and had to be verified by direct methods. First of all it was necessary to prove the existence in RNA poly- nucleotide chains of adjacent comp- lementary regions (i.e. RNA sequence information was needed).

The first known tRNA sequence was determined soon after by Holly and co-workers 2s and, importantly, could be folded into three different second-

T I B S 1 4 - D e c e m b e r 1989 507

ary structures in perfect correlat ion with the principles discussed above. One of these structures, the cloverleaf, was proved to be a good approximat ion of real secondary structure of any t R N A . The only new feature of R N A chain folding that was not predicted with the general model appeared to be the base-pairing of the 5 '- ~.nd 3 ' -end proximal segments of t R N A . The authors of the general model assumed from ' en t ropy considerat ions ' that interactions between distant .sections of R N A chain would be unlikely 18. They simply did not take into account the possibility of bringing together distant regions due to successive base-pairing of adjacent R N A segments. It was later shown that the complemen~:ary inter- actions between terminal regions is a quite c o m m o n proper ty of R N A sec- ondary structure.

Even more cogent a rguments for the original general model of R N A sec- ondary structure were accumula ted in early 1970s when some bacter iophage R N A 29 and large f ragments of ribo- somal R N A 3° were sequenced. All the types of looped-ou t structures pre- dicted by D o t y and co-workers were found in their double-helical regions. In addit ion, it was shown that G . U pairs together with classical W a t s o n - Crick pairs part icipate in format ion of R N A helical structures. Con tempor - aneously, the predict ion rules of the quanti tat ive stability of different el- ements of R N A secondary structure were developed 3~. They are based on the statistical analysis of parameters of many synthetic ol igonucleotides. Besides complemen ta ry base pairing these rules also take into co t sideration stacking interactions of neighboring nucleotide residues. That is why, for the nucleot ide sequence shown on Fig. 3, the pat tern of the secondary structure obta ined using these rules is, quite dif- ferent f rom that originally proposed. Recent versions of these rules were used to develop the compute r pro- grams which, in combina t ion with phylogenet ic compar isons of different R N A sequences and various exper- imental approaches , give very impress- ive results. Suffice it to say that now the secondary structures of any type of r ibosomal R N A can be regarded as proven (see Ref. 32 for references) .

Unfor tuna te ly , the elucidation of the three-dimensional s tructure of R N A is not so optimistic. The classical works of Alex Rich 's 33 and A a r o n Klug 's 34 labs on X-ray crysta l lography of t R N A brought ext remely impor tan t infor-

mat ion of the nature of tert iary contacts in R N A molecules (such as novel base pairs, base triples and numer- ous hyd rogen -bonded interact ions be tween bases and phosphodies te r backbone) . However , t R N A is the only R N A molecule of which the crystalline structure has been studied with high resolution and it is not known to what extent the principles of its spatial organizat ion can be applied to o ther types of R N A .

Concluding remarks I was lucky to spend my pos tdoctora l

years in the laboratories of Alexander Spirin and Paul Doty . The study of the principles of organizat ion of R N A macromolecu la r s tructure had been comple ted not long before. Being aware (from my own experience) of the very high prestige of both labs in the scientific communi ty , I of ten asked myself why this outs tanding work had not been appraised at its t rue worth. I now believe that the model was ahead of its time. Indeed , the great dis- coveries in molecular biology of the 1960s which i l luminated R N A majo r functions, did not need the knowledge of their real spatial organizat ion. The only feature of R N A structure that had to be considered was the single- s t randedness. Scientists who studied the mechanism of gene expression were not obliged to analyse m R N A second- ary structures until the a t tenuat ion of transcript ion was discovered 35. Until very recently molecular biologists con- t inued to deal only with the two- dimensional R N A World.

It is easy to predict that the situation will change in the near future. Indeed , using only a two-dimensional descrip- tion of R N A macromolecu la r s tructure it is impossible to unders tand the nature of R N A enzymat ic activities. Wi thout knowledge of R N A spatial organizat ion one cannot explain the p redominan t role of R N A in r ibosome function, the mechanisms of R N A splicing and R N A editing and o ther impor tant events in The R N A World.

Acknowledgements I am indebted to Drs H. Boedtker ,

P. D o t y and A. S. Spirin for allowing me to test their memories .

References 1 Altmann, R. (1889) Arch. Anat. Physiol.,

524-536 2 Brachet, J. (1987) Trends" Biochem. Sci. 12,

244-246

3 Doty, P. (1961) The Harvey Lectures 55, 103-139

4 Spirin, A. S. (1960) J. Mol. Biol. 2,436-446 5 Witkowski, J. 11988) Trends Biotechnol. 6,

234-243 6 Gilbert, W. (1986) Nature 319,618 7 Kirby, K. S. (1956) Biochem. J. 64,405-4/18 8 Gierer, A. and Schramm, G. (1956) Nature

177,702-703 9 Fraenkel-Conrat, H., Singer, B. and Wil-

liams, R. C. (1956) Biochim. Biophys. Acta 25, 87-96

10 Gierer, A. (1957) Nature 179, 1297-1299 11 Gomatos, P. J. and Tamm, J. (1963) Proc.

Natl Acad. Sci. USA 50,878-875 12 Rich, A. and Davies, D. R. (1956) J. Am.

Chem. Soc. 78, 3548-3549 13 Hall, B. D. and Dory, P. (1958) in Microso-

mal Particles and Protein Synthesis (Roberts, R. B., ed.), pp. 27-35, Pergamon

14 Rice, S. A. and Doty, P. (1957) J. Am. Chem. Soc. 79, 3937-3947

15 Boedtker, H. (1959) Biochim. Biophys. Acta 32,519-531

16 Spirin, A. S., Gavrilova, L. P. and Belozcrsky, A. N. (1959) Doklady Akad. Nauk SSSR 125, 65~661

17 Spirin, A. S., Gavrilova, L. P., Bresler, S. E. and Mosevitsky, M. I. 11959) Biokhimiya 24, 938-947

18 Fresco, J. R., Alberts, B. M. and Doty, P. (1960) Nature 188, 98~1t)1

19 Gavrilova, L. P., Spirin, A. S. and Belozersky, A. N. (1959) Doklady Akad. Nauk SSSR 126, 1121-1124

20 Haschemeyer, R., Singer, B. and Fraenkel- Conrat, H. (1959) Proc. Natl Acad. Sci. USA 45,313-319

21 Kisselev, N. A., Gavrilova, L. P. and Spirin, A.S. (1961)J. Mol. Biol. 3,778 783

22 Hogland, M. B. (1960) in The Nucleic Acids, Vol Ill (Chargaff, E. and Davidson, J. N., eds), pp. 349-408, Academic Press

23 Spirin, A. S. (1961) Biokhimiya26, 511-522 24 Boedtker, H., Moller, W. and Klemperer, E.

(1962) Nature 194,444-449 25 Rubin, G. M. (1973) J. Biol. Chem. 248,

3860-3875 26 Boer, P. H. and Gray, M. W. (1988) Cell55,

399-4 11 27 Fraenkel-Conrat, H. and Williams, R. C.

(1955) Proc. Natl Acad. Sci. USA 41,69(/-698 28 Holley, R. W., Apgar, J., Everett, G. A.,

Madison, J. T., Marquisee, M., Merrill, S. H., Penswick, J. R. and Zamir, A. (1965) Science 147, 1462-1465

29 Min Jou, W., Haegeman, G., Ysebaert, M. and Fie rs, W. (1972) Nature 237, 82-88

30 Fellner, P. (1974) in Ribosomes (Nomura, M., Tissieres, A. and Lengyel, P., eds) pp. 161-169, Cold Spring Harbor Laboratory

3l Tinoco, I., Uhlenbeck, O. C. and Levine, M. D. ( 1971) Nature 230,363-367

32 Noller, H. F. (1984)Annu. Rev. Biochem. 53, 119-162

33 Kin, S. H., Suddath, F. L., Quigley, G. J., McPherson, A., Sussman, J. L., Wang, A. H-J., Seeman, N. C. and Rich, A. (1974) Science 185,435-44(t

34 Robertus, J. D., Ladner, J. E., Finch, J. T., Rhodes, D., Brown, R. S., Clark, B. F. C. and Klug, A. (1974) Nature 250,546-551

35 Jackson, E. N. and Yanofsky, C. (1973) J. Mol. Biol. 76,89-101