[10] Conformation of tRNA and interaction with polynucleotide phosphorylase

106

TRANSFER RNA

[ 10]

10 mM cacodylate buffer (pH 7.0) containing 0.15 M KCI, 5 mM magnesium acetate, and 0.5 mM EDTA 13 is measured at 20 ° with a Beckman DU spectrophotometer equipped with a heating unit. Subsequently, the solution is heated at 50 ° for 10 minutes (conversion of form II to form I), and A260 is measured under the same conditions (see G. Felsenfeld, Vol. XII [119]). VISCOSITY, OPTICAL ROTATION, AND CIRCULAR DICHROISM. Other physical methods, such as viscosity, optical rotation, and circular dichroism have been employed to study tRNA conformation? ~ Other Species of t R N A

Under conditions similar to those described above, tryptophan tRNA from Salmonella typhimurium, Shigella dysenteriae, Aerobacter aerogenes, and Pseudomonas aeruginosa can be made to exist in two distinct conformations as demonstrated by amino acid acceptor capacity and MAK column chromatography; however, in Bacillus cereus, Bacillus subtilis, Serratia marcescens, and Micrococcus lysodeikticus, the form II conformation of tryptophan tRNA was not observed. Histidine tRNA from E. coli loses 40% of its activity in the absence of Mg2+; however, only 10-20% loss of amino acid acceptor activity of alanine, aspartic acid, asparagine, cystine, glutamine, glutamic acid, leucine, and valine tRNA of E. coli was observed after heat treatment in the absence of Mg2+.~5 Fresco et al. 6 reported that glutamine, histidine, tryptophan, gtutamic acid, and, possibly, leucine tRNA of E. coli, and leucine and arginine tRNA of yeast are partially inactivated in many conventional tRNA preparations. Furthermore, Fresco et al. 7 reported that species of yeast alanine, glutamine, lysine, serine, and tryptophan tRNA are partially or completely inactivated in dilute buffer without Mg 2+ at 0°; the inactive tRNA are renatured by heat treatment at 30 ° in the presence of Mg 2+ prior to assay at 0 °. Thus, detection of two distinct conformations of other species of tRNA may require conditions in which less energy is available for interconversion of native and denatured forms.

[ 10] Conformation of tRNA and Interaction with Polynucleotide Phosphorylase By M.

N. THANG, B. BELTCHEV, and M. GRUNBERG-MANAGO

Primary sequences of numerous specific tRNA's are now known. Models for secondary structures, such as the cloverleaf, 1 as well as for ~R. W. Holley, J. Apgar, G. A. Everett, J. T. Madison, M. Marquisee, S. H. Merrill, J. R. Penswick, and A. Zamir, Science, 147, 1462 (1965).

[ 10]

CONFORMATION OF tRNA

107

the tertiary structures (for an account, see reference 2) have been suggested. In addition, two conformational states of a tRNA (native and denatured) having the same primary sequence have been demonstrated by their biological functional activities (e.g., yeast leucine tRNAa and Escherichia coli tryptophan tRNA)? '4 Studies on the interaction between proteins and nucleic acids, using tRNA as a model for the mechanism of polynucleotide phosphorylase, and the enzyme as a probe for the structural analysis of tRNA, have increased our insight into some subtle differences in the configuration of tRNA in solution. In fact, polynucleotide phosphorylase is an exonuclease with a well-defined mechanism of action in the phosphorolysis reaction: i.e., the enzyme degrades a polynucleotide (or RNA) chain, in the presence of an excess of phosphate, from the 3'-OH end in a sequential way up to the terminal dinucleotide, without dissociating from the substrate. 5 This is tile so-called "processive" or "single-chain" mechanism. Thus, when a population of molecules is phosphorolyzed by polynucleotide phosphorylase, there are, at any given time, intact molecules, molecules~in the process of being degraded, and completely degraded molecules already converted into nucleotides. In the case of" tRNA, only one part of the population is degraded by the enzyme at low temperature. This is not a preferential attack on certain species tRNA since the phenomenon is observed with a population of purified tRNA with a sole sequence. The molecules resistant to polynucleotide phosphorylase attack are called R forms and the molecules susceptible to enzyme degradation are called S forms. R and S each represent a class of multiple conformational states. It is important to emphasize that we do not identify the R and the S forms with the native and denatured conformations. More detailed discussions on this subject are to be found in other articles? -7 Reagents

Polynucleotide Phosphoryl~e. The enzyme was purified from E. coli B. The purest possible preparation should be used in order to avoid 2M. Levitt, Nature (London), 224, 759 (1969); H. G. Zachau, Angew. Chem. Int. Ed. Engl. 8, 711 (1969). 3T. Lindahl, A. Adams, andJ. R. Fresco, Proc. Nat. Acad. Sci. U. S., 55, 941 (1966). 4W..I, Gartland, and N. Sueoka, Proc. Nat. Acad. Sci. U. S., 55,948 (1966). 5M. N. Thang, W. Guschlbauer, H. G. Zachau, and M. Grunberg-Manago, J. Mol. Biol. 26, 403 (1967). 6M. Grunberg-Manago, M. Cohn, M. N. Thang, B. Beltchev, A. Danchin, and L. Dimitrijevic, Proc. 4th FEBS Meeting, Osto 1967, "Structure and fun~ion of transfer RNA and 5S-RNA", Academic Press, p. 113 (1968). TM. N. Thang, B. Beltchev, and M. Grunberg-Manago, Eur. J. Biochem. in press (1970); B. Beltchev, M. N. Thang and C. Portier Eur. J. Biochem. in press (1970).

108

TRANSFER RNA

[ 10]

unreproducible results due to contamination from degradative enzymes or other proteins that interfere in the reaction between RNA and polynucleotide phosphorylase. Thus, the enzyme after the last purification step according to Williams and Grunberg-Manago s (Sephadex G-200 step), was further purified on a sucrose gradient when contaminant proteins were present. Phosphorolysis Mixture. The phosphorolysis of tRNA was performed as follows: The incubation mixture (total volume 1 ml) contains Tris-HC1 (pH 8), 50 mM; MgC12, 0.5 raM; PO4, 10 mM (either labeled with 32p or unlabeled); tRNA, A260= 5.0; E. coli polynucleotide phosphorylase, 25-40 units/ml (1 unit is defined as 1 /xmole of ADP release by phosphorolysis of poly (A) per hour at 37 °. Kinetic Studies. Aliquots (usuaUy 50-100 /xl) were withdrawn from the incubation mixture, 500/~1 HC104 2.5% were added to stop the reaction and to precipitate the enzyme and remaining tRNA. After centrifugation, 400 txl of the supernatant were taken for measurement of incorporation of 32p in the nucleoside diphosphate released. For this purpose two methods were applied. The first consists in absorbing the labeled nucleoside diphosphate from the 400 Ix. mixture onto charcoal in 1% HC104 solution; the charcoal is then filtered on nitrocellulose filter, washed several times with 1% HCIO4, then with 0.1% HC104, dried under infrared, and counted with a thin-window Geiger counter. The second method, based on the solubility of the phosphomolybdate complex in isobutanol, consists in forming this complex by adding to the 400/xl mixture 700/xl of 1.4 N H 2 S O 4 and 500/xl of 5% ammonium molybdate. After mixing, 2 ml of isobutanol is added and stirred vigorously (e.g., by means of a Vortex). Once the aqueous phase is settled, the superphase of isobutanol containing all the inorganic radioactive phosphate is aspirated. Traces of remaining isobutanol are removed by 2 ml of ether. This procedure does not change the volume of the aqueous phase. A 200-/xl quantity of the aqueous solution is dried on an aluminum planchet and counted with a thin-window Geiger counter. Both techniques give the same results. Unfractionated tRNA. Escherichia coli total transfer RNA was bought from General Biochemicals. Yeast total tRNA came from Boehringer (Germany). These samples were systematically filtered over a Sephadex G-100 column in the phosphorolysis buffer (unless otherwise specified) in order to separate any RNA's other than tRNA, such as ribosomal RNA, 5 S RNA, or aggregates of tRNA that could be present or eventuSF. R. Williams, and M. Grunberg-Manago, Biochim. Biophys. Acta 89, 66 (1964).

[ 10]

CONFORMATION OF tRNA

109

ally formed under phosphorolysis conditions. Only the 4 S RNA peak was used for this study. Purification of Sp¢c~,~ tRNA's, tRNA enriched with tyrosine (containing 60% of tRNA TM and 30% of tRNA ser) was prepared by passing total tRNA through a benzoylated DEAE-cellulose column, according to the method of Gillam et al2 tRNA Tyr, tRNA ser, and tRNAV~( were prepared by a combination of the methods of Gillam et al2 and of Kelmers et al., 1° with some modifications: At the end of the gradient with the benzoylated DEAE-cellulose column tRNA a'y~ and tRNA ser come out together with 1.5 M NaC1 containing 15% ethanol. This fraction was then passed on the Chromosorb column, according to the method of Kelmers et al. with the difference that instead of the Mg2+ gradient, there was a constant concentration of Mg2+ (10 mM) in both vessels of the gradient. The fraction enriched with tRNA T M (from the benzoylated DEAE-cellulose) was passed again on the Chromosorb column. There were two peaks of tRNAWhr; we used the second, which was purer, and called it tRNA~ hr. The purity of tRNA Tyr, tRNA s~r, and tRNA~hr were found to be about 95%, as calculated from the incorporation of the respective 14C-labeled amino acids when tested immediately after coming off the column. The amino acid acceptor activity decreases With the storage of tRNA at--20°C or--90°C, probably because aggregates are formed. Amino Acid Acceptor Activity of Total and Specific tRNA's. One h u n d r e d microliters of the reaction mixture contained, in mM concentrations: Tris.HC1 (pH 7.4), 100; Mg acetate, 10; fl-mercaptoethanol, 10; ATP, 4; tRNA, 1 A260 unit (0.1 unit, or less, in the case of purified tRNA's); [14C]amino acid (L-amino acid), 2; and crude aminoacyl-tRNA-synthetases from E. coli or yeast, 200 /~g of protein per milliliter. Incubation was carried out at 37 ° for 20 minutes. Fifty microliters of the reaction mixture were put on DEAE-cellulose paper (Whatman DE-81). Free amino acids were removed according to the method of Ingram and Pierce11; the spots of tRNA were cut out and counted in a liquid scintillation counter (Tri-Carb 314 EX) in toluene containing 5% (w/v) PPO and 0.3% (w/v) POPOP. Val is purified according to Yaniv and Gros TM and was a gift of tRNAcon Dr. Yaniv. tRNAyeast s~r is purified according to Zachau et al. 13 and was a 9I. Gillam, S. Millward, D. Blew, M. von Tigerstrom, E. Wimmer, and G. M. Tener,

Biochemistry 6, 3043 (1967). I°A. D. Kelmers, G. D. Novelli, and M. P. Stulberg,J. Biol. Chem. 240, 3979 (1965). 11V. M. Ingram, a n d J . G. Pierce, Biochemistry 1,580 (1962). 12M. Yaniv, and F. Gros,J. Mol. Biol. 44, 1 (1969). ~3H. G. Zachau, D. Diitting, and H. Feldman, Hoppe-Seyler's Z. Physiol. Chem. 347, 212 (1966).

110

TRANSFER RNA

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gift of Dr. Zachau, tRNAeo Phe n is purified according to Pearson and KelPhe is purchased from Boehringer. mers. TM tRNAyeast Procedure

Existence of R Forms and S Forms. When unfractionated or specific tRNA is phosphorolyzed by polynucleotide phosphorylase at low temperature, say 20°C, the liberation of nucleoside diphosphates proceeds readily at a relatively constant rate during the first phase of the reaction, then decreases rapidly to reach a plateau. The extent of phosphorolysis, defined as percent of initial RNA nucleotides concentration released as nucleoside diphosphates, represents the percentage of molecules completely degraded according to the "single-chain" mechanism of phosphorolysis already postulated. This means that the remaining tRNA is entirely resistant with the 3 ' O H terminal pA intact. Thus, the extent of phosphorolysis indicates the percentage of S forms in tRNA, at this temperature, while the remaining tRNA's are the R forms by definition. The R class molecules can be separated from free nucleotides released and from enzyme by filtration on Sephadex G-100 column. The unphosphorolyzed tRNA isolated in this way is only slightly, if at all, phosphorolyzed at the same temperature. The phenomenon is essentially the same (but not necessarily to the same extent at a given temperature for tRNA's) when phosphorolysis of purified specific tRNA's is assayed. The purified tRNA's assayed to date include: tRNA TM, tRNA set, tRNA TM, tRNA{ hr, tRNA Phe of E. coli, and tRNA s~r, and tRNA P~ from yeast. Dialysis from the incubation mixture after the plateau is reached shows that the dialyzed tRNA is not degraded either by polynucleotide phosphorylase. These results indicate that the stopping of the phosphorolysis is not due to an inhibition of enzymatic activity by the reaction products. Temperature-Dependence of the Two Forms. The first striking property derived from these observations is the apparent irreversibility of these two forms at low temperature. It seems that R form and S form are not in dynamic equilibrium. Nevertheless when the reaction is carried out at higher temperatures (~>25° C), the slope of the second phase indicates the reversibility of R forms into S forms, although, depending on the temperature, the rates are different. The second, not less striking point, is the fact that the transition of one class of conformations of each molecule into another is very rapid when the temperature of the solution is increased or decreased. The transition of R forms into S forms can thus be shown by a stepwise phosl*R. L. Pearson, and A. D. Kelmers,J. Biol. Chem. 24t, 76'7 (1966).

[ 10]

CONFORMATION OF tRNA

11 1

phorolysis: phosphorolysis to the plateau at a given temperature, then increase of the incubation mixture temperature induces a new burst of phosphorolysis with a rapid phase, followed by a plateau, and so on. In reverse, the transition of S forms to R forms can be shown when the incubation mixture is brought from a higher temperature to a lower one, and the phosphorolysis is immediately stopped. The temperature-dependence of the R and S forms of a tRNA in solution opens the possibility to estimate a critical temperature at which all the molecules of a given tRNA are in the S forms. This temperature is indicated by the complete phosphorolysis of a specific tRNA: for instance, the mean value for E. coli tRNA is 45 ° C, but is 37 ° C for yeast tRNA, and near to 50 ° C for rat liver tRNA. It is even possible to determine some sort of Tm of the tRNA configuration by plotting the extent of phosphorolysis (taken arbitrarily after 24 hours of incubation) versus temperature, as is usually done with ultraviolet absorption-temperature profile. It is of interest to find a correlation between the Tm estimated this way and the Tm of the first melting region of a tRNA. Time-Dependence of the Conversion. As has been mentioned, at low temperature, once the S forms are completely degraded (plateau of phosphorolysis, diphosphates liberated), the R forms can be separated by gel filtration or can be dialyzed over 24 hours without significant conversion to S forms (detectable by phosphorolysis). Nevertheless, the reversion of R forms to S forms is measurable when the remaining unphosphorolyzed tRNA is allowed to stand in solution for a much longer time than 24 hours. For instance, E. coli tRNA was phosphorolyzed at 20 ° C up to the plateau (28% in this experiment); the incubation mixture was then dialyzed against the phosphorolysis buffer over toluene at 4° C for 8 days (for control, a sample of tRNA without enzyme was dialyzed under the same conditions). The recovery of Azn0 showed that no degradation occurred under these conditions. After this long dialysis, the purpose of which was to eliminate the 32p and nucleotides, the remaining tRNA (R forms) and the control sample were phosphorolyzed under standard conditions with freshly added polynucleotide phosphorylase. The results showed that 20% phosphorolysis could be obtained after 24 hours' incubation with the unphosphorolyzed remainder of the first phosphorolysis, and the extent of phosphorolysis of the control sample was 26%. Consequently the R forms can indeed slowly revert to the S forms. Effect of Ions. Since polynucleotide phosphorylase requires a divalent cation for its activity, the question arises immediately as to the effect of these ions on the configuration discriminated by the enzyme.

112

TgANSFER RNA

[10]

First, the concentration of Mg2+ does not affect the extent of phosphorolysis in a range from 0.1-5 mM. The Tm ofE. coli tRNA at 0.5 and 5 mM, determined by the ultraviolet absorption-temperature profile, shows a difference of > 10°. This suggests that the secondary structure is not the primary factor controlling the R and S forms. In contrast, Mn2+ ions change the proportion of R and S forms, as compared to a solution containing Mg2+ at the same temperature. No effect of monovalent ions, such as NaCI, has been observed. In the presence of 0.5 mM Mg2+, Na + at a concentration up to 1 M does not change the extent of phosphorolysis. Denaturation Effect. Treatments such as irradiation, formaldehyde denaturation, exhaustive dialysis against water, do not increase the extent of phosphorolysis of both yeast and E. coli tRNA. In contrast, when total yeast tRNA was treated by dimethyl formamide, the phosphorolysis went to completion. But the extent of phosphorolysis was brought back to that of untreated tRNA preparations when such dimethyl formamide treated tRNA was previously "renatured" by heating at 60 ° in the presence of Mg before phosphorolysis. Complete phosphorolysis of tRNA I~u (yeast) can also be obtained by treatment with EDTA at 60 ° under conditions of "denaturation" as defined in this volume [9]. Similarly, renaturation of this denatured tRNA (with 10 mM Mg2+ at 60 °) reverses the phenomenon to the segregation of R and S forms by phosphorolysis. Addition of purine in the phosphorolysis medium leads the degradation almost to completion. One unexpected result comes from the effect of thermodenaturation at 100 °, followed by rapid cooling. As a matter of fact, a tRNA solution heated at 100 ° under phosphorolysis conditions for 2-3 minutes, then immediately cooled in an ice bath, can be phosphorolyzed, at a given t e m p e r a t u r e , to an extent twice that obtained with untreated tRNA. But one fails to get complete denaturation, tested by phosphorolysis (i.e., 100% phosphorolysis) even when ~oolirtg is performed by immersing the heated sample in liquid nitrogen. Furthermore, the additional extent of phosphorolysis of preheatch tRNA solutions is only, obtained when the tRNA is heated in the presence o f d i ~ l e n t ions, and at a temperature > 90 °. Preheating performed in the presence of EDTA, or in the presence of KC1 (0.1-1.0 M) in place of Me 2+, or at a temperature < 80 °, does not affect the extent of phosphorolysis: All these restrictions a n d the failure to obtain complete phosphorolysis after heating, still remain unexplained. The phenomenon observed does not seem d u e to some breakage inside the chain induced by the presence of Me 2+ and high temperature. Indeed, several reasons can he~pt~t,forward: First, the concentration

[ 10]

CONFORMATION OF tRNA

113

of Mge+ is too low to be effective in breaking chains at 100° for just 2-3 minutes; heating in the presence of higher concentrations of Mg 2+ (up to 5 raM) gives the same results as with 0.5 raM. Second, it is known that hydrolysis of the phosphodiester bonds catalyzed by divalent ions or by heating yields chain segments terminated by 3'-PO,. These polynucleotides are not phosphorolyzed by polynucleotide phosphorylase and are even inhibitors. Now, tRNA can be completely phosphorolyzed at low temperature if every time the phosphorolysis plateau is attained, the sample is heated to 100 °, then cooled, and the phosphorolysis is repeated. This successive heating and phosphorolysis leading to complete phosphorolysis of tRNA rules out the possibility suggesting breakages of chains under the experimental conditions described. Dilution. No aggregates of tRNA could be found under phosphorolysis conditions notwithstanding the use of several physical methods: gel filtration, zone density centrifugation, nuclear magnetic resonance measurement, and the use of a biological test: aminoacylation of the purified tRNA. Nevertheless, the extent of phosphorolysis at a given temperature can be markedly increased if the tRNA concentration is lower than A200 = 0.5 to 1.0, depending on the batches of tRNA used. This phenomenon is not understood, since no significant variation can be observed with concentrations higher than A2e0= 1.0. All the experiments for investigating the R and S forms have always been performed with concentrations of tRNA higher than A2n0= 1.0. Assay of Acylation. Off hand, there is no point in discussing biological properties of these two forms since, by definition, all experiments start with fully active RNA's, at least in the case of purified specific tRNA's. Nevertheless, it is worth recalling that the elimination of the S forms at each temperature does not change the specific activity for the charging of the specific tRNA. This suggests that the configurations of tRNA in solution, discriminated by polynucleotide phosphorylase, are not distinguished by aminoacyl-tRNA-synthetases. This is different with the tRNA conformations defined as native and denatured which are recognized by the synthetases and also differentiated by polynucleotide phosphorylase. In short, studies on the degradation of tRNA by polynucleotide phosphorylase show the existence of two classes of conformational states of tRNA in solution (either total or specific and purified) defined by their sensitivity or resistance to the enzyme. Furthermore, the recombined molecule made up with 3' and 5' halves of yeast tRNAnclPhe (Phillippsen et al. 15) still contains the structural requirement for the ~sp. Philippsen, R. Thiebe, W. Wintermeyer, and H. G. Zachau,Biochem. Biophys. Res. Comm. 33, 922 (1968).

114

TRANSFER RNA

[1 1]

recognition of the two classes of conformations by polynucleotide phosphorylase. 16 Hence, it seems that the integrity of the anticodon loop is not necessary for the discrimination of these conformations by the enzyme. Since two interconvertible conformations of tRNA, the native and the denatured states, have been found (at least for some species in E. coli as well as in yeast), these two findings call for the question of the relation between these conformations that have been shown to exist by different means. Experiments with tRNAyeast set and tRNAsLeUstmi) indicate that these tRNA's in the fully native state are phosphorolyzed in the manner observed with any other tRNA: segregation of R and S forms by the plateau of kinetic curves. Thus, it is unlikely that the R class of conformations can be assimilated to the native conformation. Other reasons have already been given elsewhere. The R and S forms have no apparent relation with the biological activity of tRNA, at least as tested in vitro. But it is hoped that such a study has some interest insofar as the conceptual aspect of the problem of macrornolecules interaction is concerned, and will contribute to insight of the properties of those molecules. 16B. Beltchev and M. N. Thang, Fed. Eur. Biochem. Soc. Lett. 11, 55 (1970).

[ 11] Crystallization of Transfer R N A By JAMESD. YOUNG and ROBERT M. BOCK Simple reproducible conditions have been developed for crystallizing transfer ribonucleic acids (tRNA) from aqueous solutions using controlled levels of ammonium sulfate or various organic solvents as precipitating agents. The nature of the tRNA, its purity and concentration, the nature and amount of polyvalent cations, temperature, pH, and the nature and amount, of precipitating agent(s), all seem to be important variables in obtaining crystals useful for X-ray diffraction studies: The method most successful in surveying the many variables for obtaining useful crystals equilibrates a small sample droplet containing the tRNA solution with vapors from a large solvent reservoir. The composition of the reservoir, and hence the droplet, can be readily varied and precisely controlled. The reservoir and the sample droplet(s) are placed in a vapor-tight chamber. The chamber should be such that the reservoir composition can be easily changed by additions from