Selective chemical methylation of yeast alanine transfer RNA

456

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 97472

SELECTIVE CHEMICAL METHYLATION OF YEAST ALANINE T R A N S F E R RNA

D O N A L D M. P O W E R S * AND R O B E R T W. H O L L E Y ' *

Section o[ Biochemistry and Molecular Biology, Biological Sciences Division, Cornell University, Ithaca, N. Y. z485o (U.S.A.) (Received A u g u s t 7th, 1972)

SUMMARY

Yeast tRNA Ala was methylated with [14C]dimethylsulfate in a reaction medium designed to stabilize its native structure. The intrinsic reactivity of the bases was deternfined to be G (I.OO) > A (o.61) > I (0.46) > C (0.40) > : > U, T, T (o) in these conditions. The initial site of reaction was adenine-59 in the T T C G loop, thus demonstrating that the N-I position of this residue is exposed in native tRNA A~a. Methylation of A-59 did not affect the acceptor activity. These results are consistent with the conformational models of Cramer and Levitt.

INTRODUCTION

Detailed knowledge of the structure of tRNA is essential to understanding how it functions in protein synthesis and cellular regulation. Limited modification with chemical reagents has been a useful method of obtaining information about the native conformation of a few tRNAs. In principle, residues that interact with other parts of the macromolecule are protected against specific chemical attacks. Yeast tRNA Aja has been studied by bromination 1, nitrous acid deamination 1'~ and carbodiimide addition 3, as well as by limited nuclease cleavage4. The results indicate that the anticodon loop and the lower portion of the hU loop are reactive, while the T T CG loop and the postulated double stranded segments of the cloverleaf configuration ~ are inert. Other purified tRNAs have shown similar patterns of reactivity with monoperphthalic acid e, kethoxaF, bisulfite 8,9, methoxyamine 1°, and 2-chloroethylaminen. These observations, together with physical and hydrodynamic evidence (see ref. I2), form the basis for the detailed conformational models of Cramer ~3 and Levitt 14. Additional reactivity data would be helpful in defining the true structure of tRNA Ala in solution. We investigated the use of a radioactive alkylating agent, [14Cldimethylsulfate, as a suitable chemical probe. The chemistry of methylation of nucleic acid components has been reviewed by Griffin16. The reaction is carried out in an aqueous medium at * To w h o m correspondence should be addressed. P r e s e n t address: L a b o r a t o r y of Biochemistry, National H e a r t and L u n g I n s t i t u t e , N. I. H., Bethesda, Md. 2OOl 4 ,U.S.A. ** P r e s e n t address: Salk I n s t i t u t e for Biological Studies, P. O. B o x 18o9, San Diego, Calif. 92112, U.S.A.

Biochim. Biophys. Acta, 287 (1972) 456-464

METHYLATION OF t R N A Ala

457

physiological temperature, pH and ionic strength in order to stabilize the tRNA in its native structure. Under these conditions methylation is limited to the base moieties le. The products are stable and are easily identified by the radioactive label. We wish to report the identification of a highly reactive adenine in the T ~ C G loop of yeast tRNA AI~ by this procedure. Methylation of this base does not affect alanine acceptor activity.

MATERIALS AND METHODS

Adenosine, guanosine, cytidine, uridine and inosine were purchased from P-L Biochemicals, St. Louis, Mo. I-Methyladenosine, I-methylinosine, 7-methylguanosine, 7-methylinosine, 3-methylcytidine, and N6-methyladenosine were from Cyclo Chemical Corp., Los Angeles, Calif. Pseudouridine was from Calbiochem, La Jolla, Calif. Nucleosides were checked for purity by thin-layer chromatography. Dimethylsulfate and 2-naphthoxyacetic acid were from Aldrich Chemical Corp., Milwaukee, Wisc. EU-14ClDimethylsulfate (I.O Ci/mole) was from Tracerlab, Waltham, Mass. EU-~H~Alanine (5.4 Ci/mmole) was from Schwarz Bioresearch, Orangeburg, N. Y. Ribonuclease T 1 (Sankyo) was from Calbiochem. All lots used in these experiments assayed negative for phosphatase and ribonuclease T, activities. Pancreatic ribonuclease, snake venom phosphodiesterase and alkaline phosphatase were from Worthington Biochemical Corp., Freehold, N.'J. Ribonuclease T 2 (ribonuclease CB) was from Calbiochem. DEAE-cellulose (0. 9 mequiv/g) was from Schleicher and Schuell Co., Keene, N. H. It was prepared for chromatography as described previously 17. Urea, A. R. grade, was from Mallinckrodt Chem. Co., St. Louis, Mo. Buffer solutions containing urea were treated with activated charcoal (Norit A) before chromatography to remove ultraviolet-absorbing impurities. Benzoylated DEAE-cellulose and naphthoxyacetyl-N-hydroxysuccinimide were synthesized according to Gillam et al. TM

Preparation o[ t R N A m~ Transfer RNA was prepared from commercial bakers' yeast (Fleischmann's) by extraction with aqueous phenol 19. Initial purification of tRNA Ala was by countercurrent distribution 2°. Further purification was by the napthoxyacetyl derivatization procedure is. Fractionation on a column of benzoylated DEAE-cellulose (1.2 cm × 220 cm) with a 1.2 1 gradient (0.3-0. 9 M NaC1) ~1 yielded a single peak of tRNA Ala which accepted 168o pmoles alanine per A~e0 unit. Assay/or alanine acceptor activity The extent of aminoacylation was measured according to Loehr and Keller 22. Nucleoside analysis Oligonucleotides were digested to nucleosides by a two-step procedure. 3oo/zl of water and 40/*g ribonuclease T 2 were added to 2-5 A 2e0 units of lyophilized oligonucleotide. The pH was adjusted to 5 if necessary. Digestion was carried out overnight at 37 °C. The mixture was adiusted to pH 7.5 with NH4HCO 3 and the following were added: Io/ag snake venom phosphodiesterase; 7/~g pancreatic ribonuclease; and 4/~g alkaline phosphatase. Digestion was continued for another 8 h and the mixBiochim. Biophys. Acta, 287 (I96g) 456-464

458

1). M. I'OWERS, R. W. H()LLF_Y

ture was lyophilized. Nucleosides were separated on cellulose thin layers (Brinkmann MN3oo, IOO #m). Two developments in solvent System A (ethanol o.I M ammonium acetate, pH 7.5) (7 : 3, v/v), in the first direction were followed by a single development in System B (isobutryic acid conc. NH4OH water, 66 : I : 33, by vol.) in the second. R r values for multiple development were determined according to StArka and H a m p l 2a. Ultraviolet-absorbing spots were excised and the cellulose was extracted three times with o.I M HC1. Spectra were recorded on a Cary 15 spectrophotometer at p H I, 7, and I i .

Radioactivity determinations Radioactivity was measured by liquid scintillation counting. Millipore filters were counted in toluene containing 4 g 2,5-dipheny loxazole (PPO) and o.I g 1,4-bis2-(5-phenyloxazolyl)benzene (M%POPOP) per 1. Counting efficiency was 62 % for 14C and 25 % for all. Aqueous 14C samples were counted in 2 parts of the PPO-Me.,POPOP-toluene solution and I part Triton X - I o o with 85 % efficiency.

RESULTS

Methylation o/components o/tRNA m~ Preliminary studies were carried out to determine the intrinsic reactivity of the t R N A bases in the conditions used to modify tRNA. The relative reactivity obtained for the nucleosides is given in Table I. The observed order of reaction was G > A > I > C > > U, ~ , T, with products m7G, mlA, m7I, and maC, respectively. Further reaction of m7G to mlmTG, mTI to mlmTI, and m l I to mlmTI was also observed at higher concentrations of dimethylsulfate. Similar results were obtained with the 2',3'-cyclic phosphates of A, G, C and U, and with the nucleoside diphosphates GpU, CpU and ApU, indicating that methylation at the base moiety is unaffected b y esterification of the ribose by phosphate (such as in tRNA). TABLEI METHYLATION OF NUCLEOSIDES WITH DIMETHYLSULFATE R e a c t i o n m i x t u r e s contained 500/,moles s o d i u m cacodylate, 20t, moles MgClz, I / z m o l e nucleoside, and i o o / , m o l e s d i m e t h y l s u l f a t e in a total v o l u m e of I.O ml. p H w a s 7.0. T e m p e r a t u r e w a s 20 °C. Methylation was followed s p e c t r o p h o t o m e t r i c a l l y b y the m e t h o d of Holiday .4. D a t a are expressed as ={=the S.E. for three determinations.

Nucleoside

Relative reactivity

Guanosine Adenosine Inosine Cytidine Uridine Pseudouridine 5-Methyluridine

i.oo :L o-o4 o.61 ~ o.oi o.47~-o.oI o.41 ~ o . o i

Methylation of tRNA m~ Highly purified t R N A Ala was methylated with [14Cldimethylsulfate in a series Biochim. Biophys. Acta, 287 (1972) 456-464

METHYLATION OF t R N A Ala

459

of timed reactions. After removal of unreacted dimethylsulfate, samples were taken to determine (I) the extent of methylation, (2) the distribution of [14Clmethyl groups, and (3) the alanine acceptor activity. The kinetics of the reaction are shown in Fig. Ib. Over a period of one hour an average of 2.5 methyl groups were incorporated per molecule of tRNA A~. The reaction rate decreased temporarily after approximately o.S methyl groups per molecule tRNA were incorporated, resulting in a biphasic time course. Fig. Ia shows similar reaction rate plateaux which were observed in pilot experiments with unfractionated yeast tRNA. These occurred at approximately 1. 5 methyl groups per molecule tRNA. The significance of these plateaux is not clear.

A 54

2 UN FRACT¢ONATED t R N A e

3

I

PURIFIED f R N A Al° 3

(3

i 40

20 Reaction

h

610

t i m e (re}n)

Fig. i. K i n e t i c s of t h e r e a c t i o n of [~4C]dimethylsulfate w i t h t R N A . A: U n f r a c t i o n a t e d y e a s t t R N A . R e a c t i o n s were carried o u t on a R a d i o m e t e r T T T l c / S B R 2 c p H s t a t . R e a c t i o n m i x t u r e s cont a i n e d iok~moles s o d i u m cacodylate, 2 o # m o l e s MgC12, IOO/,g t R N A a n d either i 8 o / ~ m o l e s (Curve i ) or 360/~moles (Curve 2) [14C]dimethylsulfate (o.5 Ci/mole) in i.o ml. p H was m a i n t a i n e d a t 7.o w i t h i M N a O H . T e m p e r a t u r e was 2o °C. R e a c t i o n s were t e r m i n a t e d b y e x t r a c t i o n w i t h ether, t R N A w a s r e c o v e r e d b y a d s o r p t i o n on a s m a l l c o l u m n of DEAE-cellulose, followed b y w a s h i n g w i t h o. 3 M NaC1 a n d elution w i t h I M NaC1. R a d i o a c t i v i t y w a s d e t e r m i n e d b y liquid s c i n t i l l a t i o n c o u n t i n g . ]3: H i g h l y purified t R N A A~a. E x p e r i m e n t s were carried o u t as described a b o v e e x c e p t r e a c t i o n m i x t u r e s c o n t a i n e d 25o/~g t R N A A~a a n d I 2 5 / m l o l e s [14C]dimethylsulfate.

Fig. 2 compares the results of a 4a-rain methylation experiment and a control :sample of tRNA Ala. A peak of radioactivity which corresponds to a new A~60 peak appears between Peaks I I and 9b, apparently at the expense of Peak 14. In the samples methylated for 5 and IO min this peak represents the only specific incorporation of [laClmethyl groups. Some secondary reactions were observed in the 4 ° and 6o-min samples.

Identification o/the methylated residue Fractions containing the radioactive peak were chromotographed again on DEAE-cellulose at pH 3 in 7 M urea. Fig. 3 shows that essentially all of the radioactivity is contained in the main A2e0 peak. The oligunocleotide was concentrated, Biochim. Biophys. Acta, 287 (I972) 4 5 6 - 4 6 4

460

D . M . POWERS, R. W. HOLLEY

24~

A

i

i

1,6'--

0.8~--

,if

8

!

i

E u

i 1.2

1200

o.8

800

100

Column

200 effluent

300

(ml)

Fig. 2. C h r o m a t o g r a p h y of ribonuclease T 1 digest of t R N A Ata on DEAE-cellulose. A: Control t R N A Al'. B: Methylated t R N A Ate. Details of m e t h y l a t i o n are given in Fig. I. Reaction w a s t e r m i n a t e d after 40 rain. Samples were desalted on S e p h a d e x G-25, m a d e u p to o.i M Tris-HC1, p H 7-5 and digested w i t h 400 units ribonuclease Tt for 2 h at 37 °C. Oligonucleotides were fract i o n a t e d on c o l u m n s of DEAE-cellulose (o.3 c m x i I o cm) in 7 M urea and 0.5 M s o d i u m acetate, p H 6, w i t h a 6oo-ml linear gradient of o to 0.4 M NaCI. Flow rates were m a i n t a i n e d at o, 4 ml/min. F r a c t i o n s of ~.25 ml were collected. A260 profiles were p l o t t e d a u t o m a t i c a l l y on a Gilford Model 2ooo s p e c t r o p h o t o m e t e r . Oligonucleotides are n u m b e r e d according to Penswick* and Eolley *. F o r identification of t h e ribonuclease T~ digest fragments, see ref. 17. R a d i o a c t i v i t y was det e r m i n e d b y liquid scintillation counting.

150o

0.6 f

~"

~,

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•

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600

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60

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2 0 ' ~ b =.S-C-G-C. ,,,, "--U.P ew'

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20

40

60 80 Fraction number

10Q

'

40

120

"/.G.~ Fig. 3. C h r o m a t o g r a p h y of m e t h y l a t e d oligonucleotide. 2. 4 A 2~0 u n i t s containing 9800 d p m were diluted and applied to a c o l u m n of DEAE-cellulose (0.3 c m X 50 cm) equilibrated w i t h o.I M s o d i u m acetate, p H 6, and 7 M urea. E l u t i o n w a s w i t h a gradient of 0-0.06 M NaC1 in 7 M u r e a and o, i M formic acid, p H 3. A z,o was plotted automatically. R a d i o a c t i v i t y was d e t e r m i n e d by liquid scintillation counting. F r a c t i o n s were x.25 ml.

Fig. 4. Site of m e t h y l a t i o n of t R N A A'a. The sequence of t R N A Ata is given in the cloverleaf configuration ~. Location of the m e t h y l a t e d residue (A-59) is indicated b y the arrow.

Biochim. Biophys. Acta, 287 (I972) 456-464

METHYLATION OF t R N A Ala

46I

desalted on a column of Sephadex G-I 5 and digested to nucleosides. The lyophilized digest was dissolved in 5 °/A water and analyzed by two-dimensional chromatography. Five ultraviolet-absorbing spots were observed. These were eluted and their ultraviolet spectra and radioactivity contents were measured. The compounds were identified by chromatographic mobility and ultraviolet spectra. These data are summarized in Table II. The radioactivity was confined to two methylated adenosines, mlA and m6A. Since m6A does not occur from direct methylation of adenosine, presumably it has arisen from mlA through a Dimroth rearrangement 25. The molar composition is uniquely consistent with the sequence mlA-U-U-C-C-G,corresponding to fragment 14 of a ribonuclease T 1 digest. The primary site of methylation is therefore A-59 in tRNA Ala. The location of this residue is shown in Fig. 4. T A B L E II IDENTIFICATION

OF

METHYLATi~D

NUCLEOSIDE

M e t h y l a t e d oligonucleotide (I.2 A z6o u n i t s , 723 o d p m ) recovered a f t e r c h r o m a t o g r a p h y on D E A E cellulose a t p H 3 (see Fig. 3) w a s d i g e s t e d to nucleosides a n d a n a l y z e d b y t h i n - l a y e r c h r o m a t o g r a p h y . Molar recoveries of nucleosides were d e t e r m i n e d b y u l t r a v i o l e t spectra; r a d i o a c t i v i t y b y liquid scintillation c o u n t i n g .

Thin-layer Compound chromatography spot I 2 3 4

Guanosine Uridine Cytidine i -Methyladenosine N e - M e t h y 1adenosine Total methylated adenosine

5 4+ 5

i

Nucleoside (nmoles)

Methyl groups (dpm)

21.8 41.2 45.5

15 io 30

7.6

2350

II.O 18.6

Molar ratios (nmoles)

Nucleoside

Methyl group

I.OO 1.89 2.09

----

7.2

0.35

o.33

3980

I2.2

o.50

0.56

633o

19.4

o.85

o.89

-

-

--

-

~oo,7

1.0

~o :~ ~a4

4o~ o

///

o.2

20 c L

•

0

i

i

i

20

1

i

i

40 Rooctlon time {mln)

[

o

~

60

Fig. 5. Loss of a l a n i n e a c c e p t o r a c t i v i t y a n d m e t h y l a t i o n of oligonucleotide i4. R e a c t i o n m i x t u r e s were t h o s e described in Fig. I. A l i q u o t s were d e s a l t e d a n d a s s a y e d to a c c e p t [aH]alanine, Initial a c c e p t o r a c t i v i t y w a s i 6 9 o pmoles/A,60 u n i t . T h e r e m a i n d e r of t h e r e a c t i o n m i x t u r e w a s digested w i t h r i b o n u c l e a s e T 1 a n d c h r o m a t o g r a p h e d as described in Fig. 2. T h e e x t e n t of m e t h y l a t i o n of oligonucleotide 14 w a s d e t e r m i n e d f r o m t h e r a d i o a c t i v i t y profile a n d t h e /~moles of t R N ~ Aj* chromatographed.

Biochim. Biophys. Acta, 287 (1972) 4 5 6 - 4 6 4

462

D. M. POWERS, R. W. HOLLEY

Melhylation o/ adenine-59 and alanine acceptor activity Alanine acceptor activity remaining after methylation was determined for each of the samples described in Fig. I. The extent of methylation of A-59 was determined for each sample from DEAE-cellulose oligonucleotide profiles as described in Fig. 2. The results of these experiments are shown in Fig. 5- Comparison of these data indicates that methylation of A-59 occurs more than twice as fast as alanine acceptor activity is lost. Therefore, A-59 is probably not involved in specific recognition of the alalfine t R N A synthetase.

DISCUSSION

Earlier studies have found the bases in the T ¥1 C G loop of t R N A AI~ to be relatively unreactive toward chemical modification. Nelson et al. 1 found that none of the pyrimidines in the loop were brominated b y N-bromosuccinimide. Brostoff and Ingram 3 failed to detect any incorporation of a water-soluble carbodiimide into the loop, where T-55, hu-56, G-58, U-6o and U-6I were potentially reactive. Other tRNAs have displayed similar inertness in this region. These results suggested that the T C G loop is buried within the three dimensional structure of the molecule. None of the earlier studies, however, provided any information about A-59. In yeast t R N A vhe, which was studied by Cramer et al. ~ using monoperphthalic acid, the analogous adenine was protected against N-oxidation by a naturally occurring N - I methyl group. The reactivity shown by A-59 toward dimethylsulfate is a clear indication that the N - I position is not involved in hydrogen bonding with another residue. Studies by Ludlum 28 on complexes of poly (A) and poly(U) demonstrated that hydrogen bond formation protects adenine against attack by dimethylsulfate. The failure of the adiacent G to react ---in spite of the nearly two-fold greater reactivity of guanine relative to adenine---provides additional evidence that G-58 is protected. All of these results taken together indicate a conformation in which all of the bases of the T ~u C G loop except A-59 (N-I) are protected b y interaction with other residues. t R N A Ala can be folded into both the Cramed 3 and Levitt 14 conformational models. The reaction of A-59 is consistent with both structures. Cramer's model features pairing between ~-56, C-57 and U-6o with bases in the hU loop, while G-58 base pairs with U-6I across the loop. This arrangement leaves A-59 in an exposed position and protects all of the other bases in the loop horn attack. One serious defect in this model is the implied assumption that t R N A conformation is stabilized solely by standard A • U and G • C base pairs. This limitation at level of tertiary structure seems unwarranted. Levitt's model postulates a hydrogen-bonded complex between A-59 (N-6, N-7) and Lg-56 (0- 4, N-3) which leaves the N - I position of A-59 open to react. T-55, 5u-56 and C-57 interact with bases in the hU loop, while G-58 is protected at N- 7 by a complex with A-46. U-6o and U-6I are exposed, however, which seems inconsistent with the bromination ~ and carbodiimide addition a results. Certainly neither model is entirely correct in all respects. Dimethylsulfate, as well as other reagents, is specific for certain positions in a given base. Interpretation of reactivity data must take this into account, since other positions in a reactive base m a y well be involved in structure formation at the secondary and tertiary levels. The potential influence of such interactions on the reaction Biochim. Biophys. Acta, 287 (1972) 456-464

METHYLATION OF

t R N A Ala

463

being used as a probe should also be considered. Hydrogen bonding elsewhere in the ring, e. g. perhaps the N-6 and N-7 positions of A-59 as suggested by Levitt 14, would probably affect the nucleophilic character of position N-I. So might stacking interactions with adjacent residues~L While our data clearly show that the N-I position of A-59 is open and reactive, its reactivity m a y have been enhanced because of activation b y elements ot the coniormation. The fact that acceptor activity is not lost at the same rate as A-59 is methylated suggests that this base does not have a role in the recognition of alanyl-tRNA synthetase. Eventual loss of activity m a y be due to reaction of critical bases at the synthetase recognition site, methylation of the terminal adenosine and destruction of essential elements of the conformation as the reaction progresses 28. The results demonstrate that dimethylsulfate can be used as an effective structure probe. The availability of a variety of chemical probes with different specificities is important for designing, testing, and evaluating hypothetical structure models for individual tRNAs.

ACKNOWLEDGEMENTS

This work was supported b y grants from the National Science Foundation and the National Institutes of Health. We wish to thank Mrs Indu Meshri for skillful technical assistance during the countercurrent fractionation of tRNA. REFERENCES x 2 3 4 5 6 7 8 9 IO Ii 12 13 I4 15 16 17 18 19 20 21

Nelson, J. A., Ristow, S. C. and Holley, R. W. (1967) Biochim. Biophys. Acta 149, 590-593 May, M. S. and Holley, R. W. (I97o) J. Mol. Biol. 52, 19-36 Brostoff, S. W. and Ingrain, V. M. (i97o) Biochemistry 9, 2372-2376 13enswick, J. R. and Holley, R. W. (1965) Proc. Natl. Acad. Sci. U.S. 53, 543-546 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 Cramer, F., Doepner, H., yon der Haar, F., Schlimme, E. and Seidel, H. (1968) Proc. Natl. Acad. Sci. U.S. 6i, 1384-1391 Litt, M. (1969) Biochemistry 8, 3249-3253 Kudan, ~., Freude, K. A., Kudan, I. and Chambers, R. W. (1971) Nature, New Biol. 232, 177-179 Singhal, R. 13. (1971) J. Biol. Chem. 246, 5848-5851 Cashmore, T. (1971) Nature, New Biol. 230, 236-239 Vlasov, V. V., Grineva, N. I. and t
464 22 23 24 25 26 27 28

D.M. POWERS, R. W. HOLLEY

Loehr, J. s. and Keller, E. B. (1968) Proc. Natl. Acad. Sci., U.S. 61, I i i 5 - i i 2 2 StArka, L. and Hampl, R. (1963) J. Chromatogr. 12, 347-351 Holiday, E. R. (1936) Biochem. J. 3o, 1795-18o 3 Macon, J. B. and Wolfenden, R. (1968) Biochemistry 7, 3453-3458 Ludlum, D. B. (1965) Biochim. Biophys. Acta 95, 674-676 Fuller, W. and Hodgson, A. (1967) Nature 215, 817-821 ~ern~, J., Rychltk, I. and norm, F. (1966) Coll. Czech. Chem. Commun. 31,336--345

Biochim. Biophys. Acta, 287 (1972) 456-464