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Circular dichroism of valine and formylmethionine transfer RNA from Escherichia coli: Effect of aminoacylation
BIOCHIMICA ET BIOPHYSICA ACTA
I83
BBA 96411
CIRCULAR DICHROISM OF VALINE AND FORMYLMETHIONINE TRANSFER RNA FROM E S C H E R I C H I A COLI: EFFECT OF AMINOACYLATION ALICE J. A D L E R AND GERALD D. FASMAN
Graduate Department o] Biochemistry, Brandeis University, Waltham, Mass. (U.S.A.)" (Received September I9th, 1969)
SUMMARY
The ultraviolet circular dichroism spectra of two specific tRNA's, valine and formylmethionine-I, from Escherichia coli have been examined under various conditions. No significant changes were observed upon aminoacylation of either tRNA. Limits were placed upon possible differences in conformation (at most two basepair hydrogen bonds and less than ten percent change in base stacking interactions) between the acylated and deacylated molecules. Differences are reported between valine and formylmethionine tRNA's. The circular dichroism of each tRNA species changes as a function of pH.
INTRODUCTION
There has recently been much interest in elucidating the conformation of specific tRNA molecules. This problem is being attacked by X-ray, physico-chemical, and chemical modification studies (for example, see refs. 1-3, respectively) resulting usually in refined cloverleaf models4. A related question is whether tRNA undergoes any structural change upon aminoacylation. Such a change would help explain the observed increase in binding to ribosomes of charged tRNA 5. GANTT et al. s, have recently approached this problem by means of hydrogen exchange, and have found a small, but probably significant, increase in structure upon aminoacylation of mixed Escherichia coli tRNA; three or four extra hydrogens per molecule became stabilized. Optical rotatory methods have given conflicting results: SARIN AND ZAMECNIK7 observed an 18 % decrease in the 26o-nm Cotton effect amplitude of the optical rotatory dispersion (ORD) when E. coli tRNA was charged. On the other hand, HASHIZUMEAND IMAHORI8 reported that there was no significant change in the circular dichroism (CD) peak at 265 nm upon aminoacylation of yeast tRNA. (The apparent 5 % increase was within experimental error.) It should be emphasized that these previous experiments were performed on unfractionated mixtures of tRNA's. However, each specific acceptor tRNA has a somewhat different primary and secondary structure. This uniqueness is Abbreviations: CD, circular dichroism; ORD, optical rotatory dispersion. * This paper is publication No. 684 of this department.
Biochim. Biophys. Acta, 204 (197 o) 183-19o
184
A . J . ADLER, G. D. FASMAN
reflected in a slightly different ORD pattern for each tRNA which has been studied 9,1° (yeast aspartic acid, glycine, lysine, alanine and tyrosine). Several additional papers have been concerned with the CD 11-13 and ORD 18-17 of non-acylated tRNA under various conditions, and relevant inferences about structure have been made. Therefore, the present investigation utilized two isolated E. coli tRNA's, valine and formylmethionine-i, and examined their CD spectra in the stripped and loaded ( > 50 %) states. Neither species showed any significant conformational change upon charging.
MATERIALS AND METHODS
Materials Unfractionated E. coli B tRNA was obtained from Schwarz. The isolated samples of E. coli B valine and formylmethionine-I tRNA's (lot IA) were obtained from Dr. A. D. Kelmers (see ref. 18) through a National Institute of General Medical Sciences program. Any attached amino acids were removed 19 with 0.5 M Tris (pH 8.8) from all tRNA samples, which were then dialyzed against o.oi M Tris, o.oi M MgC12 (pH 7.3). Purified extracts of E. eoli aminoacyl tRNA synthetases, prepared in the manner of YAMANE AND SUEOKA19, were the gift of Dr. K. Moldave. Radioactive amino acids, L-I3Hjvaline (generally labelled, 2340 #C/#mole) and L-[Me-3Hlmethio nine (specific activity 135/,C//~mole) were from New England Nuclear. ATP was supplied by P-L Chemicals, reduced glutathione by Sigma, and unlabelled amino acids by Nutritional Biochemicals. Salts, buffers, and phenol were reagent grade. t R N A aminoacylation Charging was carried out by the method of YAMANE AND SUEOKA 19, with some modifications taken from WEISS et al. is. Synthetase concentrations were such that further increase in enzyme concentration did not raise the yield of aminoacylation. (Preliminary experiments to determine saturating enzyme levels were performed with unfractionated tRNA; these results were checked with the purified tRNA's.) Charging reactions were carried out for 25 min at 37°; the pH was 7.3. For valine the reaction mixture contained I.O mg tRNA val, 1.2 mg protein (in the synthetase extract), reagents (in #moles): [3H]valine 0.34 , Tris 250, ATP 5, MgC12 25, glutathione IO, KC1 25, in 2.5-ml volume. The methionine mix contained 0.2 mg tRNA 1~Met, o. 4 m g protein, reagents (#moles): E3H]methionine i.o, Tris ioo, ATP 5, MgC12 Io, glutathione 4, KC1 IO, in i.o ml. The tRNA's were isolated, after aminoacylation, by phenol extraction (80 ~o phenol adjusted to pH 4.6) and ethanol precipitation2°; the procedure was modified to include two washes of the tRNA precipitates with an aqueous solution containing 67 O//o ethanol and 0.6 °/o potassium acetate at pH 4.6. The purification procedure did not remove any radioactive amino acids. (The original preparation of the specific tRNA's (ref. 18) contained a phenol step. Furthermore, tRNA val and tRNA ~Met are not among the renaturable tRNA's of E. coli 21. Therefore, the present phenol extraction should cause no conformational changes.) The charged tRNA samples, as well as portions of the non-acylated tRNA's, were dialyzed against (and stored frozen in) 0.02 M sodium acetate (pH 4.6), o.oi M magnesium acetate. This treatment ensured that all tRNA samples would be dissolved in the same buffer, for CD measureBiochim. Biophys. Acta, 204 (i97 o) i83-19o
CIRCULAR DICHROISM OF t R N A
185
ments. After aminoacylation (and after storage for one month) the t R N A T M was 55 % charged, the tRNA[ Met 50 ~o. There was quantitative recovery of the RNA. Aminoacylation was monitored b y means of a Nuclear Chicago scintillation counter. Samples were usually precipitated with 5 % trichloroacetic acid onto Whatman glass fiber paper; purified aminoacyl-tRNA's at low p H yielded the same results when deposited directly onto the paper. Aliquots of amino acids were counted in the presence of appropriate amounts of unlabelled tRNA, to avoid quenching effects. Readings were always greater than 800 counts/min.
Concentration determination Concentrations of t R N A solutions used for CD were measured spectrophotometrically, b y means of a Cary 14 spectrophotometer, in the 25o-27o-nm region. Extinction coefficients were determined b y phosphate assay ~. Absorption m a x i m a were always at 258 nm. The values for e (for a given solvent) were independent (to ± 0 . 5 % ) of t R N A species and of aminoacylation state. Extinction coefficients used, on a nucleotide residue basis, are 7.5O.lO 3 l m o l e - l . c m -~ in o.I M Tris, o.oi M MgC12 (pH 7.3), and 7.96. lO 3 in 0.02 M acetate, o.oi M magnesium acetate (pH 4.6). This corresponds to 23.4 absorbance units for I mg t R N A per ml at p H 7 in a I-cm cell. values were 2 °/o higher in the absence of magnesium. Circular dichroism CD measurements were performed with a Cary 6o spectropolarimeter equipped with a 6OOl CD attachment. The wavelength range was 200-320 nm. Fused quartz, jacketted cells (I m m or I cm) from Optical Cell Co. were used; concentrations were such that the m a x i m u m absorbance was about one. CD results are reported in terms of r~l (residue ellipticity), which has units of deg.cm2.dmole -1. Signals at maior peaks were larger than 0.02 degrees. Experimental errors (due largely to the CD noise level) are given in Table I. Temperatures below ambient were maintained with a Tamson refrigerated circulating bath. Curve resolving The CD curves were analyzed into their component peaks b y means of a DuPont 31o curve resolver.
RESULTS
The CD data taken at p H 4.6 are shown in Fig. I. Major peak ellipticity values are tabulated in Table I. At this p H no decrease in amino acid label was detected after several hours at room temperature. For each specific t R N A (tRNA T M and t R N A f~et) the state of aminoacylation is seen to have no significant effect upon the CD spectra; that is, all differences are within the experimental precision. (Limits will later be set upon any possible conformational changes consistent with the data.) In one experiment an amount of free amino acid (valine or methionine) equivalent to the concentration of uncharged t R N A molecules was added. The purpose of this experiment was to see whether a possible conformational change upon aminoacylation could be obscured b y the CD signal of the amino acid present. The added Biochim. B~ophys. Acta, 204 (197o) 183-19o
0
I
oo
0
4~
4.6 4.6 4.6
7.3 7-3 7.3
7.0 7.o 7.3
7-3
Formylmethionine Formylmethionine Formylmethionine
Valine Valine Valine
Formylmethionine Formylmethionine Formylmethionine
unfractionated
uncharged
charged uncharged (above sample, deacylated) uncharged (fresh sample)
charged uncharged (above sample, deacylated) uncharged (fresh sample)
charged uncharged uncharged, with added m e t
charged uncharged uncharged, with added valine
State o/aminoacylation
* E x p e r i m e n t a l error ! o . 3 × lO 3. *" E r r o r ~ o . 5. lO3. *** E r r o r ± 1 . 1 o 3 at p H 4.6, zk3.IO~ at p H 7-3-
4.6 4.6 4.6
pH
Valine Valine Valine
Specific t R N A
296.5
296. 5 296 296
297 297 296.5
296 296 296
295.5 295.5 295-5
2t
--I.I
--1.6 --1.7 --2.0
--o.9 --i.i --1. 3
--2.0 --2. 4 --2. 3
-- 1.5 -- 1.3 --i.6
[011"
265
266. 5 266.5 266. 5
266.5 266. 5 267
267 267 267. 5
267 267 267
22
+22. 3
+24.2
+24.1 +23.9
+21.1 +2o.4 +21.6
+2o. 5 +20.8 +21.o
+ 19.8 + 20. 5 +20. 7
I032"*
21o
21o 211.5 21o
21o 211.5 211
21o 211 212
21o 211 211
~3
--22. 5
--25.o --25.8 --31.o
--29.2 --29.5 --30.9
--26.o
--28. 5
--28. 4
28.I -- 28. 7 --28.9
[013"*"
Only well-defined b a n d s are tabulated. W a v e l e n g t h s are r e p o r t e d in nm, and are precise to ± 0 . 5 nm. Residue ellipticity values are all × lO -3. T e m p e r a t u r e s and buffer compositions are found in the figures.
CIRCULAR DICHROISM BAND VALUES
TABLE I
~q
t3 t~ 0~
-7,:
CIRCULAR I)ICHROISM OF t R N A
187 30
30
r
I
I
I
I
I
I
I
20
20
10
? o
_0
/
-I0
-i0
-20
-20
-30
-30
-40 200
t
- J
I
I
220
240
260
280
~- (rim)
I 300
-40 320
I 200
220
I 340
I 260
I 280
I 300
320
~ (rim)
Fig. i. Circular dichroism of tRNA's at pH 4.6. - - , tRNAVal, filled symbols; - - -, tRNAfMet, open symbols. Circles indicate aminoacylated samples; triangles, uncharged RNA; squares, uncharged RNA plus an added amount of the corresponding free amino acid (valine or methionine) equal to the molar concentration of RNA. Solvent: o.o2 M sodium acetate, o.oi M magnesium acetate, pFI 4.6. Temperature 23°. Concentrations approx, io -a M nucleotide residues. Path length i mm. Vertical bars show experimental uncertainty intrinsic in the measurements. Fig. 2. Circular dichroism of tRNA's at pH 7.3, tRNAVal, filled symbols; - - -, tRNAfMet, open symbols. Circles, aminoacylated RNA; triangles, same samples after deacylation at 37°; squares, fresh samples of uncharged RNA. Crosses indicate uncharged, unfractionated RNA. Solvent: o.1 M Tris-HC1, o.oi M MgC1v pH 7.3 (except pH 7.o for (S) and & data). Temperature 5°. Concentrations approx, lO-4 M nucleotide residues. Path length i cm. a m i n o acids caused no noticeable differences in CD spectra and, therefore, a m i n o acid ellipticity could n o t be complicating the results. CD d a t a were also o b t a i n e d at p H 7.3 (Fig. 2 a n d Table I). A t this more physiological pH, the nucleotide bases carry no charges, which could possibly affect the CD of t R N A . (This is in c o n t r a s t to the s i t u a t i o n at p H 4.6.) I n addition, at p H 7.3 a t R N A c o n c e n t r a t i o n of a b o u t lO -4 M residues was chosen (Io-fold lower t h a n the c o n c e n t r a t i o n at p H 4.6). A t this lower concentration, there was no d a n g e r of association of the t R N A molecules to form dimers in the presence of o.oi M m a g n e s i u m .3. The solutions of a m i n o a c y l a t e d t R N A ' s were m a i n t a i n e d at 0-5 ° d u r i n g p r e p a r a t i o n of the solutions (by a d d i t i o n of o.I M Tris to the stock solutions in dilute acetate buffer) a n d d u r i n g the CD runs. (As a control, similar precautions were t a k e n with the u n l o a d e d t R N A samples.) D u r i n g these procedures, less t h a n 3 % of the radioa c t i v i t y was removed. After these CD spectra were taken, the p H 7.3 solutions of a m i n o a c y l a t e d t R N A T M a n d t R N A fiet were set at 37 ° for 5 h allowing hydrolysis; more t h a n 98 % loss of a m i n o acid label resulted. These d e a c y l a t e d solutions were t h e n cooled to 5 ° for CD experiments. As can be seen from Fig. 2, for each t R N A species the three sets of d a t a (charged, deacylated, a n d uncharged) coincide, t h u s
Biochim. Biophys. Acta, 204 (197o) 183-19o
I88
A . J . ADLER, G. D. FASMAN
confirming the results of the p H 4.6 experiments that there is no consistent change in CD upon aminoacylation. The CD spectrum of mixed t R N A was taken in order to point out differences among the t R N A species. All the original CD spectra were analyzed into their component ellipticity bands by means of a curve resolver. An example of this analysis, for tRNA wl at p H 4.6, is given in Fig. 3. The curves were shown to consist of positive bands at
30
20
.= =S
g
Q.
I0
0
2\ -I0
i
-20
-30
-40 170
I
I
I
I
200
230
260
290
320
(rim)
Fig. 3- Analysis of CD d a t a for tRNAVal at p H 4.6. E x p e r i m e n t a l and s u m m e d simulated curves, --~. I n d i v i d u a l resolved peaks (I t h r o u g h 6) dashed and d o t t e d lines. All channels of the curve resolver were set for Gaussian hands. The d a t a for all tRNAVal samples (aminoacylated or not) could be fitted to the same simulated curve. The wavelengths, relative heights, and percentages of total curve area for each resolved b a n d are as follows: B a n d I, 199 nm, + 11. 4, io ~o; B a n d 2, 21o. 5 nm, --14.5, 33 o/. /o, B a n d 3, 227 rim, - - I . I , 2 %; B a n d 4, 235 nm, --2.9, 8 %; B a n d 5, 266.5 nnl, + 9 . 8 , 41 ~o; B a n d 6, 292 n m , - I.O, 3.5 %. The relative positions and m a g n i t u d e s of B a n d s I and 2 are s o m e w h a t arbitrary,
approximately 266 and 2oo nm (the latter band is needed to account for the shape of the 21o nm band), and negative bands at about 294, 234, 227 and 21o nm. For any given t R N A species and pH, e.g., t R N A vai at p H 7.3, all the data could be made to fit the same simulated curve within the noise level regardless of state of charging. The coincidence of data was particularly good above 215 nm (where the signal-tonoise ratio was good). Thus, curve resolving yields additional evidence that aminoacylation of t R N A causes no significant conformational change, as determined by CD.
DISCUSSION
Limits upon possible structural dijjerences caused by charging In view of previous reports of possible increases ~'s or decreases 7 in t R N A second a r y structures upon aminoacylation, it is of interest to examine the present data for any trends which might indicate conformational changes. The CD peak at about 267 nm is chosen for scrutiny since nearly all experimental measurements and structural interpretations have relied upon this band (and its ORD counterpart). InvestiBiochim. Biophys. Acta, 2o4 (197 o) 183-19o
CIRCULAR DICHROISM OF t R N A
189
gators agree 1°,13,17 that a red shift of this band, coupled to a small decrease in magnitude, indicates the breaking of hydrogen bonds between base pairs. On the other hand, a decrease in amplitude, with no wavelength change, points to the unstacking of adjacent bases. Table I shows that at pH 7-3 the values of E6~]~67n m for each tRNA species are identical to :k2 %, with no trends apparent upon aminoacylation; this is within the experimental uncertainty of ! 2 . 5 %. tRNA TM may undergo a red shift of ~< 0.5 nm upon deacylation, although this is probably within experimental error. If the sample were IOO % (instead of 55 %) charged, this would correspond to a shift of I nm. Depending on the data analysis employed, this shift indicates there may be one 1° to three 17 additional hydrogen bonds in the charged form of tRNA va~. No such shift is apparent at pH 7.3 for tRNA ~Met. At pH 4.6, tRNA ~Metmay exhibit a shift similar to the one just analyzed for tRNA wl at pH 7-3. In addition, at pH 4.6 E6~],e7nm for the acylated species may be 4 % lower for tRNA v~L and 2 °/o lower for tRNA fMet. The tRNA wl difference may be slightly outside of experimental error, and may indicate a breaking of 6 to IO °/o of stacking interactions in the charged form, from comparison with heat denaturation data 11. The slight decrease in E6)~2e7nm for tRNA TM indicates that the aminoacylated form has less ordered structure. This is difficult to reconcile with the wavelength shift data, which shows an increase in order. Probably neither inference is significant. In any case, only small, if any, structural changes are compatible with the data for valine and formylmethionine-I tRNA's. This result is consistent with findings of GANTT et al. e and of HASHIZUME AND IMAHORI8, who saw possible small increases in hydrogen bonding and in base stacking, respectively, upon aminoacylation of mixed tRNA. The results are not consistent with the data of SARIN AND ZAMECNIK 7, which imply that charged tRNA has 15-3o ~o fewer bases stacked 1° than does uncharged. However, these data 7 were measured in the absence of magnesium, where denaturation is a possibility TM. Circular dichroism per se can detect only those changes in structure which alter the asymmetry near chromophoric groups. The present CD results do not exclude the possibility of changes in tertiary folding upon aminoacylation, provided that the secondary structure (base pairing and stacking) remains essentially unperturbed. Such differences in tertiary structure might affect molecular shape and be detectable by other methods. This might explain the changes in chromatographic properties** and sedimentation velocity~5 found for several tRNA's upon aminoacylation. Dependence of CD on p H and on t R N A species The differences in CD spectra caused by changes in pH and by different acceptor tRNA's are much larger than those caused b y aminoacylation. Some effects of pH (ref. 7) and acceptor type 9,1° have been noted in ORD spectra. Both tRNA TM and tRNA fMetundergo a small red shift (approx. 0.5 nm) of the major CD band when the pH is lowered from 7.3 to 4.6; this shift probably reflects partial protonation of adenosine and cytidine residues. The slight decreases in E@]2e: nm at pH 4.6 (especially for tRNA fMet) may be caused by the higher temperature of measurement. At pH 4.6, only slight differences are apparent between the two species: the Biochim. Biophys. Mcta, 2o 4 (197o) 183-19o
19o
A . J . ADLER, G. D. FASMAN
magnitude of the small, negative band at 296 nm is larger for tRNA ~Met, and the 267rim band is narrower. At pH 7.3, additional differences appear: tRNA fMet has a larger [012,7 ~m than tRNA val, a smaller E6)]210n m and totally lacks the small negative resolved band at 227 nm (see Fig. 3 for an example of this band). Mixed tRNA has a slightly different CD spectrum. These variations are attributable to base composition or conformational differences among the various acceptor tRNA molecules, and are independent of aminoacylation. ACKNOWLEDGEMENTS
We would like to thank the National Institute of General Medical Sciences and Dr. A. D. Kelmers (Oak Ridge) for the isolated tRNA samples, Dr. K. Moldave (University of Pittsburgh) for the synthetase preparation, and Drs. L. Grossman (Brandeis) and M. Simpson (Stony Brook) for helpful discussions. This work was supported by research grants form the National Science Foundation Grant No. GB 8642, the National Institutes of Health Grant No. AM-o5852, American Heart Association Grant No. 69739, and National Institutes of Health Institutional Grant No. 7044-03 • REFERENCES I ]3. P. DOCTOR, W. FULLER AND N. L. Wt~BB, Nature, 221 (1969) 5 8. 2 I. C. P. SMITH, T. YAMANE AND R. G. SHULMAN, Can. J. Biochem., 47 (1969) 480. 3 F. CRAMER, H. DOEPNER, V. VAN DER HAAR, E. SCHLIMME AND H. SEIDEL, Proc. Natl. Acad. Sci. U.S., 61 (I968) 1384 . 4 R. W. HOLLEY, J. APGAR, G. A. EVERETT, J. T. MADISON, ~V[. MARQUISEE, S. i~I. MERRILL J. R. PENSWlCK AND A. ZAMIR, Science, 147 (1965) 1462. 5 N. W. SEEDS, J. A. RETSEMA AND T. W. CONWAY, J. Mol. Biol., 27 (1967) 421. 6 R. R. GANTT, S. W. ENGLANDER AND M. V. SIMPSON, Biochemistry, 8 (1969) 475. 7 P. S. SARIN AND P. C. ZAMECNIK, Biochem. Biophys. Res. Commun., 20 (1965) 400. 8 H. HASHIZUME AND K. IMAHORI, J. Biochem. Tokyo, 61 (1967) 738. 9 P. S. SARIN, P. C. ZAMECNIK, P. L. ]DERGQUIST AND J. F. SCOTT, Proc. Natl. Acad. Sci. U.S., 55 (1966) 579. IO J. 1~. VOURNAKIS AND H. A. SCHERAGA, Biochemistry, 5 (1966) 2997. I i J. BRAHMS AND W. F. H. M. MOMMAERTS, J. Mol. Biol., IO (1964) 73. 12 T. OHTA, I. SHIMADA AND K. IMAHORI, J. Mol. Biol., 26 (1967) 519. 13 A. ADAMS, T. LINDAHL AND J. R. FRESCO, Proe. Natl. Acad. Sci. U.S., 57 (1967) 1684. 14 G. D. FASMAN, C. LINDBLOW ANn E. SEAMAN,J. Mol. Biol., 12 (1965) 630. 15 M. R. LAMBORG AND P. C. ZAMECNIK, Biochem. Biophys. Res. Commun., 20 (1965) 328. 16 M. R. LAMBORG, P. C. ZAMECNIK, T.-K. LI, J. Ki4GI AND ]3. L. VALLEE, Biochemistry, 4 (1965) 63. 17 C. R. CANTOR, S. R. JASKUNAS AND I. TINOCO, JR., J. Mol. Biol., 20 (1966) 39. 18 J. F. WEISS, 1~. L. PEARSON AND A. D. KELMERS, Biochemistry, 7 (1968) 3479. 19 T. YAMANE AND N. SUEOKA, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) IO93. 20 K. MOLDAVE, Methods Enzymol., 6 (1963) 757. 21 T. LINDAHL, A. ADAMS AND J. R. FRESCO, Proc. Natl. Acad. Sci. U.S., 55 (1966) 941. 22 ]3. N. AMES AND D. T. DUBIN, J. Biol. Chem., 235 (196o) 769 . 23 D. ]3. MILLAR AND R. F. STEINER, Biochemistry, 5 (1966) 2289. 24 R. STERN, L. E. ZUTRA AND U. Z. LITTAUER, Biochemistry, 8 (1969) 313 . 25 H. KAJI AND Y. TANAKA, Biochim. Biophys. Acta, 138 (1967) 642.
Biochim. Biophys. Acta, 204 (197 o) 183-19o
I83
BBA 96411
CIRCULAR DICHROISM OF VALINE AND FORMYLMETHIONINE TRANSFER RNA FROM E S C H E R I C H I A COLI: EFFECT OF AMINOACYLATION ALICE J. A D L E R AND GERALD D. FASMAN
Graduate Department o] Biochemistry, Brandeis University, Waltham, Mass. (U.S.A.)" (Received September I9th, 1969)
SUMMARY
The ultraviolet circular dichroism spectra of two specific tRNA's, valine and formylmethionine-I, from Escherichia coli have been examined under various conditions. No significant changes were observed upon aminoacylation of either tRNA. Limits were placed upon possible differences in conformation (at most two basepair hydrogen bonds and less than ten percent change in base stacking interactions) between the acylated and deacylated molecules. Differences are reported between valine and formylmethionine tRNA's. The circular dichroism of each tRNA species changes as a function of pH.
INTRODUCTION
There has recently been much interest in elucidating the conformation of specific tRNA molecules. This problem is being attacked by X-ray, physico-chemical, and chemical modification studies (for example, see refs. 1-3, respectively) resulting usually in refined cloverleaf models4. A related question is whether tRNA undergoes any structural change upon aminoacylation. Such a change would help explain the observed increase in binding to ribosomes of charged tRNA 5. GANTT et al. s, have recently approached this problem by means of hydrogen exchange, and have found a small, but probably significant, increase in structure upon aminoacylation of mixed Escherichia coli tRNA; three or four extra hydrogens per molecule became stabilized. Optical rotatory methods have given conflicting results: SARIN AND ZAMECNIK7 observed an 18 % decrease in the 26o-nm Cotton effect amplitude of the optical rotatory dispersion (ORD) when E. coli tRNA was charged. On the other hand, HASHIZUMEAND IMAHORI8 reported that there was no significant change in the circular dichroism (CD) peak at 265 nm upon aminoacylation of yeast tRNA. (The apparent 5 % increase was within experimental error.) It should be emphasized that these previous experiments were performed on unfractionated mixtures of tRNA's. However, each specific acceptor tRNA has a somewhat different primary and secondary structure. This uniqueness is Abbreviations: CD, circular dichroism; ORD, optical rotatory dispersion. * This paper is publication No. 684 of this department.
Biochim. Biophys. Acta, 204 (197 o) 183-19o
184
A . J . ADLER, G. D. FASMAN
reflected in a slightly different ORD pattern for each tRNA which has been studied 9,1° (yeast aspartic acid, glycine, lysine, alanine and tyrosine). Several additional papers have been concerned with the CD 11-13 and ORD 18-17 of non-acylated tRNA under various conditions, and relevant inferences about structure have been made. Therefore, the present investigation utilized two isolated E. coli tRNA's, valine and formylmethionine-i, and examined their CD spectra in the stripped and loaded ( > 50 %) states. Neither species showed any significant conformational change upon charging.
MATERIALS AND METHODS
Materials Unfractionated E. coli B tRNA was obtained from Schwarz. The isolated samples of E. coli B valine and formylmethionine-I tRNA's (lot IA) were obtained from Dr. A. D. Kelmers (see ref. 18) through a National Institute of General Medical Sciences program. Any attached amino acids were removed 19 with 0.5 M Tris (pH 8.8) from all tRNA samples, which were then dialyzed against o.oi M Tris, o.oi M MgC12 (pH 7.3). Purified extracts of E. eoli aminoacyl tRNA synthetases, prepared in the manner of YAMANE AND SUEOKA19, were the gift of Dr. K. Moldave. Radioactive amino acids, L-I3Hjvaline (generally labelled, 2340 #C/#mole) and L-[Me-3Hlmethio nine (specific activity 135/,C//~mole) were from New England Nuclear. ATP was supplied by P-L Chemicals, reduced glutathione by Sigma, and unlabelled amino acids by Nutritional Biochemicals. Salts, buffers, and phenol were reagent grade. t R N A aminoacylation Charging was carried out by the method of YAMANE AND SUEOKA 19, with some modifications taken from WEISS et al. is. Synthetase concentrations were such that further increase in enzyme concentration did not raise the yield of aminoacylation. (Preliminary experiments to determine saturating enzyme levels were performed with unfractionated tRNA; these results were checked with the purified tRNA's.) Charging reactions were carried out for 25 min at 37°; the pH was 7.3. For valine the reaction mixture contained I.O mg tRNA val, 1.2 mg protein (in the synthetase extract), reagents (in #moles): [3H]valine 0.34 , Tris 250, ATP 5, MgC12 25, glutathione IO, KC1 25, in 2.5-ml volume. The methionine mix contained 0.2 mg tRNA 1~Met, o. 4 m g protein, reagents (#moles): E3H]methionine i.o, Tris ioo, ATP 5, MgC12 Io, glutathione 4, KC1 IO, in i.o ml. The tRNA's were isolated, after aminoacylation, by phenol extraction (80 ~o phenol adjusted to pH 4.6) and ethanol precipitation2°; the procedure was modified to include two washes of the tRNA precipitates with an aqueous solution containing 67 O//o ethanol and 0.6 °/o potassium acetate at pH 4.6. The purification procedure did not remove any radioactive amino acids. (The original preparation of the specific tRNA's (ref. 18) contained a phenol step. Furthermore, tRNA val and tRNA ~Met are not among the renaturable tRNA's of E. coli 21. Therefore, the present phenol extraction should cause no conformational changes.) The charged tRNA samples, as well as portions of the non-acylated tRNA's, were dialyzed against (and stored frozen in) 0.02 M sodium acetate (pH 4.6), o.oi M magnesium acetate. This treatment ensured that all tRNA samples would be dissolved in the same buffer, for CD measureBiochim. Biophys. Acta, 204 (i97 o) i83-19o
CIRCULAR DICHROISM OF t R N A
185
ments. After aminoacylation (and after storage for one month) the t R N A T M was 55 % charged, the tRNA[ Met 50 ~o. There was quantitative recovery of the RNA. Aminoacylation was monitored b y means of a Nuclear Chicago scintillation counter. Samples were usually precipitated with 5 % trichloroacetic acid onto Whatman glass fiber paper; purified aminoacyl-tRNA's at low p H yielded the same results when deposited directly onto the paper. Aliquots of amino acids were counted in the presence of appropriate amounts of unlabelled tRNA, to avoid quenching effects. Readings were always greater than 800 counts/min.
Concentration determination Concentrations of t R N A solutions used for CD were measured spectrophotometrically, b y means of a Cary 14 spectrophotometer, in the 25o-27o-nm region. Extinction coefficients were determined b y phosphate assay ~. Absorption m a x i m a were always at 258 nm. The values for e (for a given solvent) were independent (to ± 0 . 5 % ) of t R N A species and of aminoacylation state. Extinction coefficients used, on a nucleotide residue basis, are 7.5O.lO 3 l m o l e - l . c m -~ in o.I M Tris, o.oi M MgC12 (pH 7.3), and 7.96. lO 3 in 0.02 M acetate, o.oi M magnesium acetate (pH 4.6). This corresponds to 23.4 absorbance units for I mg t R N A per ml at p H 7 in a I-cm cell. values were 2 °/o higher in the absence of magnesium. Circular dichroism CD measurements were performed with a Cary 6o spectropolarimeter equipped with a 6OOl CD attachment. The wavelength range was 200-320 nm. Fused quartz, jacketted cells (I m m or I cm) from Optical Cell Co. were used; concentrations were such that the m a x i m u m absorbance was about one. CD results are reported in terms of r~l (residue ellipticity), which has units of deg.cm2.dmole -1. Signals at maior peaks were larger than 0.02 degrees. Experimental errors (due largely to the CD noise level) are given in Table I. Temperatures below ambient were maintained with a Tamson refrigerated circulating bath. Curve resolving The CD curves were analyzed into their component peaks b y means of a DuPont 31o curve resolver.
RESULTS
The CD data taken at p H 4.6 are shown in Fig. I. Major peak ellipticity values are tabulated in Table I. At this p H no decrease in amino acid label was detected after several hours at room temperature. For each specific t R N A (tRNA T M and t R N A f~et) the state of aminoacylation is seen to have no significant effect upon the CD spectra; that is, all differences are within the experimental precision. (Limits will later be set upon any possible conformational changes consistent with the data.) In one experiment an amount of free amino acid (valine or methionine) equivalent to the concentration of uncharged t R N A molecules was added. The purpose of this experiment was to see whether a possible conformational change upon aminoacylation could be obscured b y the CD signal of the amino acid present. The added Biochim. B~ophys. Acta, 204 (197o) 183-19o
0
I
oo
0
4~
4.6 4.6 4.6
7.3 7-3 7.3
7.0 7.o 7.3
7-3
Formylmethionine Formylmethionine Formylmethionine
Valine Valine Valine
Formylmethionine Formylmethionine Formylmethionine
unfractionated
uncharged
charged uncharged (above sample, deacylated) uncharged (fresh sample)
charged uncharged (above sample, deacylated) uncharged (fresh sample)
charged uncharged uncharged, with added m e t
charged uncharged uncharged, with added valine
State o/aminoacylation
* E x p e r i m e n t a l error ! o . 3 × lO 3. *" E r r o r ~ o . 5. lO3. *** E r r o r ± 1 . 1 o 3 at p H 4.6, zk3.IO~ at p H 7-3-
4.6 4.6 4.6
pH
Valine Valine Valine
Specific t R N A
296.5
296. 5 296 296
297 297 296.5
296 296 296
295.5 295.5 295-5
2t
--I.I
--1.6 --1.7 --2.0
--o.9 --i.i --1. 3
--2.0 --2. 4 --2. 3
-- 1.5 -- 1.3 --i.6
[011"
265
266. 5 266.5 266. 5
266.5 266. 5 267
267 267 267. 5
267 267 267
22
+22. 3
+24.2
+24.1 +23.9
+21.1 +2o.4 +21.6
+2o. 5 +20.8 +21.o
+ 19.8 + 20. 5 +20. 7
I032"*
21o
21o 211.5 21o
21o 211.5 211
21o 211 212
21o 211 211
~3
--22. 5
--25.o --25.8 --31.o
--29.2 --29.5 --30.9
--26.o
--28. 5
--28. 4
28.I -- 28. 7 --28.9
[013"*"
Only well-defined b a n d s are tabulated. W a v e l e n g t h s are r e p o r t e d in nm, and are precise to ± 0 . 5 nm. Residue ellipticity values are all × lO -3. T e m p e r a t u r e s and buffer compositions are found in the figures.
CIRCULAR DICHROISM BAND VALUES
TABLE I
~q
t3 t~ 0~
-7,:
CIRCULAR I)ICHROISM OF t R N A
187 30
30
r
I
I
I
I
I
I
I
20
20
10
? o
_0
/
-I0
-i0
-20
-20
-30
-30
-40 200
t
- J
I
I
220
240
260
280
~- (rim)
I 300
-40 320
I 200
220
I 340
I 260
I 280
I 300
320
~ (rim)
Fig. i. Circular dichroism of tRNA's at pH 4.6. - - , tRNAVal, filled symbols; - - -, tRNAfMet, open symbols. Circles indicate aminoacylated samples; triangles, uncharged RNA; squares, uncharged RNA plus an added amount of the corresponding free amino acid (valine or methionine) equal to the molar concentration of RNA. Solvent: o.o2 M sodium acetate, o.oi M magnesium acetate, pFI 4.6. Temperature 23°. Concentrations approx, io -a M nucleotide residues. Path length i mm. Vertical bars show experimental uncertainty intrinsic in the measurements. Fig. 2. Circular dichroism of tRNA's at pH 7.3, tRNAVal, filled symbols; - - -, tRNAfMet, open symbols. Circles, aminoacylated RNA; triangles, same samples after deacylation at 37°; squares, fresh samples of uncharged RNA. Crosses indicate uncharged, unfractionated RNA. Solvent: o.1 M Tris-HC1, o.oi M MgC1v pH 7.3 (except pH 7.o for (S) and & data). Temperature 5°. Concentrations approx, lO-4 M nucleotide residues. Path length i cm. a m i n o acids caused no noticeable differences in CD spectra and, therefore, a m i n o acid ellipticity could n o t be complicating the results. CD d a t a were also o b t a i n e d at p H 7.3 (Fig. 2 a n d Table I). A t this more physiological pH, the nucleotide bases carry no charges, which could possibly affect the CD of t R N A . (This is in c o n t r a s t to the s i t u a t i o n at p H 4.6.) I n addition, at p H 7.3 a t R N A c o n c e n t r a t i o n of a b o u t lO -4 M residues was chosen (Io-fold lower t h a n the c o n c e n t r a t i o n at p H 4.6). A t this lower concentration, there was no d a n g e r of association of the t R N A molecules to form dimers in the presence of o.oi M m a g n e s i u m .3. The solutions of a m i n o a c y l a t e d t R N A ' s were m a i n t a i n e d at 0-5 ° d u r i n g p r e p a r a t i o n of the solutions (by a d d i t i o n of o.I M Tris to the stock solutions in dilute acetate buffer) a n d d u r i n g the CD runs. (As a control, similar precautions were t a k e n with the u n l o a d e d t R N A samples.) D u r i n g these procedures, less t h a n 3 % of the radioa c t i v i t y was removed. After these CD spectra were taken, the p H 7.3 solutions of a m i n o a c y l a t e d t R N A T M a n d t R N A fiet were set at 37 ° for 5 h allowing hydrolysis; more t h a n 98 % loss of a m i n o acid label resulted. These d e a c y l a t e d solutions were t h e n cooled to 5 ° for CD experiments. As can be seen from Fig. 2, for each t R N A species the three sets of d a t a (charged, deacylated, a n d uncharged) coincide, t h u s
Biochim. Biophys. Acta, 204 (197o) 183-19o
I88
A . J . ADLER, G. D. FASMAN
confirming the results of the p H 4.6 experiments that there is no consistent change in CD upon aminoacylation. The CD spectrum of mixed t R N A was taken in order to point out differences among the t R N A species. All the original CD spectra were analyzed into their component ellipticity bands by means of a curve resolver. An example of this analysis, for tRNA wl at p H 4.6, is given in Fig. 3. The curves were shown to consist of positive bands at
30
20
.= =S
g
Q.
I0
0
2\ -I0
i
-20
-30
-40 170
I
I
I
I
200
230
260
290
320
(rim)
Fig. 3- Analysis of CD d a t a for tRNAVal at p H 4.6. E x p e r i m e n t a l and s u m m e d simulated curves, --~. I n d i v i d u a l resolved peaks (I t h r o u g h 6) dashed and d o t t e d lines. All channels of the curve resolver were set for Gaussian hands. The d a t a for all tRNAVal samples (aminoacylated or not) could be fitted to the same simulated curve. The wavelengths, relative heights, and percentages of total curve area for each resolved b a n d are as follows: B a n d I, 199 nm, + 11. 4, io ~o; B a n d 2, 21o. 5 nm, --14.5, 33 o/. /o, B a n d 3, 227 rim, - - I . I , 2 %; B a n d 4, 235 nm, --2.9, 8 %; B a n d 5, 266.5 nnl, + 9 . 8 , 41 ~o; B a n d 6, 292 n m , - I.O, 3.5 %. The relative positions and m a g n i t u d e s of B a n d s I and 2 are s o m e w h a t arbitrary,
approximately 266 and 2oo nm (the latter band is needed to account for the shape of the 21o nm band), and negative bands at about 294, 234, 227 and 21o nm. For any given t R N A species and pH, e.g., t R N A vai at p H 7.3, all the data could be made to fit the same simulated curve within the noise level regardless of state of charging. The coincidence of data was particularly good above 215 nm (where the signal-tonoise ratio was good). Thus, curve resolving yields additional evidence that aminoacylation of t R N A causes no significant conformational change, as determined by CD.
DISCUSSION
Limits upon possible structural dijjerences caused by charging In view of previous reports of possible increases ~'s or decreases 7 in t R N A second a r y structures upon aminoacylation, it is of interest to examine the present data for any trends which might indicate conformational changes. The CD peak at about 267 nm is chosen for scrutiny since nearly all experimental measurements and structural interpretations have relied upon this band (and its ORD counterpart). InvestiBiochim. Biophys. Acta, 2o4 (197 o) 183-19o
CIRCULAR DICHROISM OF t R N A
189
gators agree 1°,13,17 that a red shift of this band, coupled to a small decrease in magnitude, indicates the breaking of hydrogen bonds between base pairs. On the other hand, a decrease in amplitude, with no wavelength change, points to the unstacking of adjacent bases. Table I shows that at pH 7-3 the values of E6~]~67n m for each tRNA species are identical to :k2 %, with no trends apparent upon aminoacylation; this is within the experimental uncertainty of ! 2 . 5 %. tRNA TM may undergo a red shift of ~< 0.5 nm upon deacylation, although this is probably within experimental error. If the sample were IOO % (instead of 55 %) charged, this would correspond to a shift of I nm. Depending on the data analysis employed, this shift indicates there may be one 1° to three 17 additional hydrogen bonds in the charged form of tRNA va~. No such shift is apparent at pH 7.3 for tRNA ~Met. At pH 4.6, tRNA ~Metmay exhibit a shift similar to the one just analyzed for tRNA wl at pH 7-3. In addition, at pH 4.6 E6~],e7nm for the acylated species may be 4 % lower for tRNA v~L and 2 °/o lower for tRNA fMet. The tRNA wl difference may be slightly outside of experimental error, and may indicate a breaking of 6 to IO °/o of stacking interactions in the charged form, from comparison with heat denaturation data 11. The slight decrease in E6)~2e7nm for tRNA TM indicates that the aminoacylated form has less ordered structure. This is difficult to reconcile with the wavelength shift data, which shows an increase in order. Probably neither inference is significant. In any case, only small, if any, structural changes are compatible with the data for valine and formylmethionine-I tRNA's. This result is consistent with findings of GANTT et al. e and of HASHIZUME AND IMAHORI8, who saw possible small increases in hydrogen bonding and in base stacking, respectively, upon aminoacylation of mixed tRNA. The results are not consistent with the data of SARIN AND ZAMECNIK 7, which imply that charged tRNA has 15-3o ~o fewer bases stacked 1° than does uncharged. However, these data 7 were measured in the absence of magnesium, where denaturation is a possibility TM. Circular dichroism per se can detect only those changes in structure which alter the asymmetry near chromophoric groups. The present CD results do not exclude the possibility of changes in tertiary folding upon aminoacylation, provided that the secondary structure (base pairing and stacking) remains essentially unperturbed. Such differences in tertiary structure might affect molecular shape and be detectable by other methods. This might explain the changes in chromatographic properties** and sedimentation velocity~5 found for several tRNA's upon aminoacylation. Dependence of CD on p H and on t R N A species The differences in CD spectra caused by changes in pH and by different acceptor tRNA's are much larger than those caused b y aminoacylation. Some effects of pH (ref. 7) and acceptor type 9,1° have been noted in ORD spectra. Both tRNA TM and tRNA fMetundergo a small red shift (approx. 0.5 nm) of the major CD band when the pH is lowered from 7.3 to 4.6; this shift probably reflects partial protonation of adenosine and cytidine residues. The slight decreases in E@]2e: nm at pH 4.6 (especially for tRNA fMet) may be caused by the higher temperature of measurement. At pH 4.6, only slight differences are apparent between the two species: the Biochim. Biophys. Mcta, 2o 4 (197o) 183-19o
19o
A . J . ADLER, G. D. FASMAN
magnitude of the small, negative band at 296 nm is larger for tRNA ~Met, and the 267rim band is narrower. At pH 7.3, additional differences appear: tRNA fMet has a larger [012,7 ~m than tRNA val, a smaller E6)]210n m and totally lacks the small negative resolved band at 227 nm (see Fig. 3 for an example of this band). Mixed tRNA has a slightly different CD spectrum. These variations are attributable to base composition or conformational differences among the various acceptor tRNA molecules, and are independent of aminoacylation. ACKNOWLEDGEMENTS
We would like to thank the National Institute of General Medical Sciences and Dr. A. D. Kelmers (Oak Ridge) for the isolated tRNA samples, Dr. K. Moldave (University of Pittsburgh) for the synthetase preparation, and Drs. L. Grossman (Brandeis) and M. Simpson (Stony Brook) for helpful discussions. This work was supported by research grants form the National Science Foundation Grant No. GB 8642, the National Institutes of Health Grant No. AM-o5852, American Heart Association Grant No. 69739, and National Institutes of Health Institutional Grant No. 7044-03 • REFERENCES I ]3. P. DOCTOR, W. FULLER AND N. L. Wt~BB, Nature, 221 (1969) 5 8. 2 I. C. P. SMITH, T. YAMANE AND R. G. SHULMAN, Can. J. Biochem., 47 (1969) 480. 3 F. CRAMER, H. DOEPNER, V. VAN DER HAAR, E. SCHLIMME AND H. SEIDEL, Proc. Natl. Acad. Sci. U.S., 61 (I968) 1384 . 4 R. W. HOLLEY, J. APGAR, G. A. EVERETT, J. T. MADISON, ~V[. MARQUISEE, S. i~I. MERRILL J. R. PENSWlCK AND A. ZAMIR, Science, 147 (1965) 1462. 5 N. W. SEEDS, J. A. RETSEMA AND T. W. CONWAY, J. Mol. Biol., 27 (1967) 421. 6 R. R. GANTT, S. W. ENGLANDER AND M. V. SIMPSON, Biochemistry, 8 (1969) 475. 7 P. S. SARIN AND P. C. ZAMECNIK, Biochem. Biophys. Res. Commun., 20 (1965) 400. 8 H. HASHIZUME AND K. IMAHORI, J. Biochem. Tokyo, 61 (1967) 738. 9 P. S. SARIN, P. C. ZAMECNIK, P. L. ]DERGQUIST AND J. F. SCOTT, Proc. Natl. Acad. Sci. U.S., 55 (1966) 579. IO J. 1~. VOURNAKIS AND H. A. SCHERAGA, Biochemistry, 5 (1966) 2997. I i J. BRAHMS AND W. F. H. M. MOMMAERTS, J. Mol. Biol., IO (1964) 73. 12 T. OHTA, I. SHIMADA AND K. IMAHORI, J. Mol. Biol., 26 (1967) 519. 13 A. ADAMS, T. LINDAHL AND J. R. FRESCO, Proe. Natl. Acad. Sci. U.S., 57 (1967) 1684. 14 G. D. FASMAN, C. LINDBLOW ANn E. SEAMAN,J. Mol. Biol., 12 (1965) 630. 15 M. R. LAMBORG AND P. C. ZAMECNIK, Biochem. Biophys. Res. Commun., 20 (1965) 328. 16 M. R. LAMBORG, P. C. ZAMECNIK, T.-K. LI, J. Ki4GI AND ]3. L. VALLEE, Biochemistry, 4 (1965) 63. 17 C. R. CANTOR, S. R. JASKUNAS AND I. TINOCO, JR., J. Mol. Biol., 20 (1966) 39. 18 J. F. WEISS, 1~. L. PEARSON AND A. D. KELMERS, Biochemistry, 7 (1968) 3479. 19 T. YAMANE AND N. SUEOKA, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) IO93. 20 K. MOLDAVE, Methods Enzymol., 6 (1963) 757. 21 T. LINDAHL, A. ADAMS AND J. R. FRESCO, Proc. Natl. Acad. Sci. U.S., 55 (1966) 941. 22 ]3. N. AMES AND D. T. DUBIN, J. Biol. Chem., 235 (196o) 769 . 23 D. ]3. MILLAR AND R. F. STEINER, Biochemistry, 5 (1966) 2289. 24 R. STERN, L. E. ZUTRA AND U. Z. LITTAUER, Biochemistry, 8 (1969) 313 . 25 H. KAJI AND Y. TANAKA, Biochim. Biophys. Acta, 138 (1967) 642.
Biochim. Biophys. Acta, 204 (197 o) 183-19o