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Tertiary structure determinants in transfer RNA
32
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96064
T E R T I A R Y S T R U C T U R E D E T E R M I N A N T S IN T R A N S F E R RNA I. P S E U D O U R I D I N E "
DAVID B. M I L L A R
Laboratory of Physical Bio6hemistry, Naval Medical Research Institute, National Naval Medical Center, Bethesda, Md. (U.S.A.) (Received J u l y 5th, 1968)
SUMMARY
The structural perturbation effects resulting from the cyanoethylation of Escherichia coli transfer RNA (tRNA) have been studied. The results suggest that: I. The C-C-A terminus area in cyanoethylated tRNA is in a more exposed state than in native tRNA. 2. Cyanoethylated t R N A is more susceptible to nuclease degradation than native. 3. There is a loss of A - U base pairs in cyanoethylated tRNA. 4. Cyanoethylated t R N A is capable of forming a compact tertiary conformation in Mg ~÷ but it is a different one than that obtaining for native tRNA. 5. Cyanoethylated t R N A does not show the same Mg 2+ induced cooperative absorbance melting profile as native tRNA. This is probably due to the altered tertiary conformation.
INTRODUCTION
The concept of tertiary structure in transfer RNA (tRNA) arose because of the observation that during the helix-coil transition of t R N A there were temperature scale divergencies between optical and hydrodynamic data. This appears to be the case in yeast and Escherichia coli t R N A (refs. I, 2) in neutral salt and in E. coli t R N A in the presence of Mg *+ (ref. 2) although for yeast t R N A Mg 2+ it has been suggested 1 that the thermally induced collapse of the secondary and tertiary structure occurs simultaneously. While the secondary structure of E. coli t R N A appears to be generated and largely stabilized by hydrogen-bonding determinants, base stacking and other secondary interactions among the four common bases 3, the determinants of the tertiary conformation are at present unknown. A common feature of the tRNA's whose primary structure is known is that looped-out areas are a direct consequence of arranging the bases into a maximally * F r o m Bureau of Medicine and Surgery, N a v y D e p a r t m e n t , Reseach t a s k MRoo5.o6.ooo4. The opinions in this paper are those of the a u t h o r and do n o t necessarily reflect tke views of t h e N a v y D e p a r t m e n t or the Naval Service at large.
Biochim. Biophys. Acta, i74 (1969) 32-42
TERTIARY STRUCTURE DETERMINANTS IN
tRNA
33
hydrogen-bonded hypothetical secondary structure. Viewed in this manner, the looped-out bases do not contribute directly to the stability of the secondary structure. However, if the helical limbs are folded in a highly restricted manner such that two* or more limbs come into close approximation, interaction between the residues of the looped-out areas becomes possible. The biological expression of tRNA requires a discrete tertiary conformation 1 and such discretion is likely to come from base pairs. If this suggestion is correct, such base pairs become "primary determinants" of tertiary structure and other non-covalent stabilizing interactions become "secondary determinants". As possible candidates for the role of primary determinants there is, a priori, no reason to discard any of the bases in the looped-out areas (saving the anti-codon and bases unable to hydrogen bond). Recent work on the selective modification of pseudouridine residues and accompanying loss of biological activity offers a means to check the possibility of this residue playing a role in the maintenance of the tertiary structure of tRNA. Conditions for the modification of pseudouridine vary considerably 5-8 and solvent conditions play a major role in directing the extent as well as the rate of reaction 9,1°. In our study, the conditions of RAKE AND TENER 1° have been used to cyanoethylate all the pseudouridine residues in tRNA. The choice to cyanoethylate all the residues was made on the basis of the work of YOSHIDAAND U K I T A 9'11 who demonstrated that in the presence of 0. 5 M NaC1 only about 1.2 mole % of the pseudouridine reacted and there were no changes in the melting curves of the modified tRNA.
EXPERIMENTAL
Cyanoethylation of pseudouridine was accomplished by following the procedure described in RAKE AND TENER 1°. Under the solvent conditions chosen (50 % dimethyl sulfoxide, 50 % water, 9 ° min at 37 °) only pseudouridine is the major reactant; less than I mole of uridine/mole t R N A reacts. It was found that rigid precautions against contaminating ribonuclease were necessary. Cyanoethylated t R N A was purified by 2-fold icecold ethanol precipitation (in 0.2 M KC1) and dialysis against water at 4 ° for 18 h. Following this, the cyanoethylated t R N A was placed on a G-75 Sephadex column and eluted with water. The central two-thirds of the peak was taken. It was found that during water elution chromatography of unmodified t R N A on Sephadex various cuts of the eluate did not all display the same degree of aggregation towards Mg 2+ as did bulk tRNA. The aggregation tendency decreased as the cuts progressed from the emerging front to the tailing edge. This m a y indicate that all species of t R N A do not aggregate as readily as others. This finding is under investigation. Cyanoethy]ated t R N A was concentrated by pervaporation, and then lyophilized. The degree of cyanoethylation was followed using the spectrophotometric data of CHAMBERS 6, OFENGAND 12, and the nuclease digestion conditions of * T h e a p p r o x i m a t i o n m u s t be a c c o m p l i s h e d b y m e a n s of specific folding o v e r o t h e r a r e a s of t h e molecule since it h a s b e e n s h o w n for E. coli t R N A (ref. 4) t h a t h i g h m o l e c u l a r w e i g h t f r a g m e n t s or m i x e s of f r a g m e n t s e q u a l l i n g t h e m o l e c u l a r w e i g h t of t R N A do n o t e x h i b i t cooperat i v e b e h a v i o r in Mg ~+, w h i c h is one of t h e d i s t i n g u i s h i n g f e a t u r e s of t h e t e r t i a r y s t r u c t u r e in t h i s solvent. H e n c e , t h e full c h a i n l e n g t h of i n t a c t t R N A is i n v o l v e d in t h e folding n e c e s s a r y to a s s u m e a tertiary conformation.
Biochim. Biophys. Acta, 174 (1969) 32-42
34
D . B . MILLAR
RAKE AND TENER1°. Nucleoside chromatography (two dimensional) was done according to OFENGAND12. Molecular weights were estimated using the YPHANTIS procedure 13. Viscosity measurements were performed as before 8. Coupling of the fluorescent dye, acriflavine, to tRNA was accomplished as outlined by MILLARAND STEINER~. A complete analysis of the apparatus and methods used to measure polarization of fluorescence and the factors affecting the measurements has been published 14. Pancreatic ribonuclease was a product of Sigma Chemical Co. Ribonuclease T-I was a produce of Calbiochem, as was snake venom phosphodiesterase (Russel's viper venom). In testing the action of nucleases on native or modified tRNA, o.3-ml aliquots of the reaction mixture were pipetted into 0.3 ml of icecold 0.25 % uranyl acetate, 4 % perchloric acid*, incubated io min in an ice bath, centrifuged for 5 min at 5 ° at 2500 rev./min in an International PR-I centrifuge; 0.3 ml of the superuatant was added to 0.7 ml of H20 and read at 260 m#. Zero-time blanks were taken. In preparing pancreatic ribonuclease digests for column chromatography, the action of ribonuclease was stopped by the addition of bentonite 4 (suspended in o.I M EDTA). The slurry was incubated at o ° for IO min and the bentonite removed by 20 min centrifugation at 17 ooo rev./min in an International Model B-2o centrifuge at 4 °. I ml of the supernatant was placed on a 50.5 cm × I.O cm column of Sephadex G-75 previously equilibrated with o.I M EDTA. The column was run at room temperature (20-23 °) and o.8-ml fractions were collected. The absorption spectrum (recorded on a Cary Model 14 M) of native and modified tRNA to two different temperatures (IO ° and 80 °) was analyzed by means of the technique of FRESCO, KLOTZ AND RICHARDS15. Absorption melting curves were done as previously described ~.
RESULTS
Number o/pseudouridine residues cyanoethylated It was estimated that 2.3 moles of pseudouridine per mole of tRNA were cyanoethylated after 9 ° rain at 37 ° in 50 % dimethylsulfoxide, 5° % H20, I.O M dimethylaminoethanol, 0.45 M acrylonitrile. E. coli tRNA contains 4-thiouridine is, which as the free base reacts with acrylonitrile 1~. The data indicated that 0. 4 mole per mole tRNA had been modified. This value is lower than that reported by OFENSAND12 and may be due to the alkaline lability of cyanoethylated thiouridine (the pH of the reaction is above II).
Homogeneity, molecular weight, sedimentation coefficient, and viscosity o/cyanoethylated t R N A A plot of In y vs. r ~ for cyanoethylated tRNA was rectilinear and the molecular weight was 25 600. The sedimentation coefficient (ultraviolet optics) of cyanoethylated tRNA was indistinguishable from that of native tRNA. The intrinsic viscosity at 20 ° in o.oi M Tris, 0.02 M Mg 2+ (pH 7.0) was determined to be 0.038 * The author is grateful to Dr. M. SINGER for suggestions concerning the precipitation procedure.
Biockim. Biophys. Acta, 174 (1969) 32-42
TERTIARY STRUCTURE DETERMINANTS IN
tRNA
35
dl/g in fairly good agreement with the value of o.o41 dl/g noted previously*. In o. 3 M KC1, o.oi M "Iris (pH 7.o), the value is 0.054 dl/g in good agreement with the value of 0.059 reported by MILLARAND MACKENZIE17. This data indicates that no gross hydrodynamic changes occurred in tRNA upon cyanoethylation and that like native tRNA, cyanoethylated tRNA is able to assume a compact configuration in the presence of Mg*+.
A bsorbance melting curves
Fig. I shows the absorbance melting curves of cyanoethylated tRNA compared to native tRNA in neutral salt and in Mgz+. In agreement with the results of RAKE AND TENERTM there is virtually no difference in thermal denaturation behavior between native and modified tRNA in neutral salt although the maximum absorbance increase is slightly less. In Mgz+, however, the melting profile of modified tRNA is less sharp and cooperative. Controls (omission of acrylonitrile in the incubation reaction) showed only a slight difference from native tRNA melting curves in magnesium.
1.30
t.25
1.20
1,15
P.IO
1.05
0
B d
1.3o
1,25
1.20
I, i5
i. lO
LOS
I 0
I0
r~
t~ 20
I 30
40
50
60
70
80
90
TEMPERATURE
Fig. I. Melting profiles of n a t i v e (C)) a n d c y a n o e t h y l a t e d t R N A ( O ) . (A) 0.02 M Mg 2+, o.oi M Tris (pH 7.o); (B) 0. 3 M KC1, o.oi M Tris (pH 7.0). I n b o t h c a s e s t h e u p p e r s e t of p o i n t s a t 20 ° i n d i c a t e s t h e reversible n a t u r e of tile profile.
Biochim. Biophys. Acta, 174 (1969) 32-42
36
D.B. MILLAR
Analysis o/ the thermal denaturation spectrum The absorbance melting profiles were analyzed by the method of FRESCO, KLOTZ AND RICHARDS15. There m a y be slightly more GC pairs melting out in native TABLE I BASE PAIRS MELTING OUT BETWEEN VARIOUS TEMPERATURES
Temperature range (°C)
Soh, ent
IO-80 lO-61 lO-8O
o.o2 M M g * + + o . o i M Tris (pH 7.o) o.o2 M M g * + + o . o I M Tris (pH 7.0) 0. 3 M K C l + o . o i M Tris (pH 7.0)
Native t R N A
Cyanoethylated t R N A
IAu*
IGC*
IAU*
/GC**
o.31
o.47
0.32
0.46
o.24 ,~o.o8 0.24
o.43 ,-~o.o8 0.45
* fAU and fGC axe the fractions of total nucleotides in AU or GC pairs, respectively.
than in cyanoethylated tRNA and there is approximately a 2o % decrease in AU pairs melted out in cyanoethylated t R N A as compared with native. In view of the high temperature, the plateauing of the absorbance curve of cyanoethylated t R N A (Fig. I) and the known temperature stability of short length AU pairs 18,x9 a reasonable interpretation of this result would be that there are less AU pairs in cyanoethylated t R N A than in native tRNA. The temperature zone between io ° and 61 ° in magnesium was also investigated since it is here that differences in the melting curve of modified t R N A vs. native t R N A are first seen. It should be pointed out that the values of fAU and fac estimated for this temperature range are very imprecise (4-20 %) due to the small amplitude of the denaturation spectra (Table I). The values indicate that there are a small number of AU and GC pairs which are noncooperatively melting out before the remainder of the AU and GC pairs in cyanoethylated tRNA.
Effect o/cyanoethylation on helical rigidity The question arises as to what effect the modification of t R N A has on helical rigidity; that is, the rotational freedom of the bases. This point was investigated by means of the experiment shown in Fig. 2 which shows a plot of I / P + I / 3 vs. Tin. Here P is a polarization of fluorescence, T is absolute temperature, and n is solvent viscosity. In this experiment, loss of internal rotational rigidity which accompanies the disruption of the ordered structure of a polynucleotide ~'14 is signaled by a drop of P, hence I / P + I / 3 rises. Comparison of this data with that for native t R N A (refs. 2, I7) shows that the thermally induced acquisition of rotational freedom is virtually the same in cyanoethylated tRNA as it is in native tRNA. This result implies that neither the base pairs melting out early nor the "lost" AU pairs in cyanoethylated t R N A (Table I) cannot all be concentrated adjacent to the C-C-A terminus where the fluorescent label is bound (on the ribose of the adenosine residue). This follows since if all the base pairs were in this area, their thermal dissociation (and increase in rotational freedom) should have been reflected by a decrease in P markedly different from native t R N A (ref. 20). Such is not the case. If, however, Bioehim. Biophys. Acta, 174 (1969) 32-42
TERTIARY STRUCTURE DETERMINANTS IN t R N A
.o}
37
TEMPERATI.RZ 75
2'
~25
42
5:
5~
6~
0.~
5O
0 I
4oh g
30) ~o~ s'
°[
,;o ~'o ~o
o'o ~'0 ,;0
,~
go
tOO
ZSO
30~
._ •
40c
:JME, M~NUTES
Fig. 2. Polarization of fluorescence thermal ~ofile of an acriflavine conjugate of cyanoethylated tRNA. O, 0.3 M KC1, o.oi M Tris (pH 7.0); 0 , 0.02 MMg *+, o.oi MTris (pH 7.o). The temperature scale is adjusted to be in register with values of T/n. Concentration approx. 0. 3 mg/ml. ~ excitation 460 m/~; emission 5IO m/z. Fig. 3. Phosphodiesterase digestion of native tRNA (O) and cyanoethylated tRNA (O). Solvent: o.i M Tris, 0.02 M Mg*+ (pH 8.o). Digestion was started by the addition of i ml of phosphodiesterase (i5o units/ml in tile above solvent) to 2 ml of a tRNA solution; final concentratiort of tRNA: o.314 mg/ml. The extent of digestion was determined as described in the text.
the bases were localized n e a r the C - C - A t e r m i n u s a n d it was i m m o b i l i z e d b y some o t h e r r o t a t i o n a l l y r e s t r a i n i n g forces (e.g., either efficient base s t a c k i n g or the i n t i m a t e p r o x i m i t y of o t h e r helical regions - - i.e., " t i g h t l y b u r i e d " within the molecule) t h e n t h e s i t u a t i o n m i g h t possibly be rationalized.
Action o/venom phosphodiesterase on modified t R N A To check t h e l a t t e r point, t h e action of v e n o m p h o s p h o d i e s t e r a s e on n a t i v e a n d c y a n o e t h y l a t e d t R N A was investigated. W i t h b o t h phosphodiesterase a n d ribonuclease digestion (reported on below), controls were g e n e r a l l y digested at i d e n t i c a l rates as n a t i v e t R N A ; a l t h o u g h in some cases there was a slight increase (approx. IO % ) in rates of digestion. I t is seen (Fig. 3) t h a t a t 37 °, t h e action of p h o s p h o d i e s t e r ase is twice as extensive on c y a n o e t h y l a t e d t R N A as on native. This indicates t h a t t h e nuclease a t t a c k proceeds p a s t t h e C - C - A m o i e t y in modified t R N A ; since in n a t i v e t R N A , nuclease a t t a c k ceases a t a b o u t this p o i n t zl (in Mg 2+ at 20°). This d a t a indicates t h e C - C - A t e r m i n u s a n d t h e i m m e d i a t e base pairs n e x t to it are not d e e p l y a n d / o r rigidly b u r i e d in t h e s t r u c t u r e of c y a n o e t h y l a t e d t R N A . A t o °, n a t i v e a n d c y a n o e t h y l a t e d t R N A are e q u a l l y a t t a c k e d b y phosphodiesterase, i n d i c a t i n g t h a t b e t w e e n 37 ° a n d o ° some e v e n t has occurred in c y a n o e t h y l a t e d t R N A which confers p r o t e c t i o n a g a i n s t t h e nuclease.
Action o/ribonuclease on cyanoethylated t R N A N a t i v e t R N A in a similar Mg 2+ c o n t a i n i n g solvent is r e l a t i v e l y r e s i s t a n t t o the action of ribonuclease 4,23.~4. A t 20 ° (Fig. 4) with c y a n o e t h y l a t e d t R N A b o t h t h e initial r a t e a n d e x t e n t of d e g r a d a t i o n are c o n s i d e r a b l y g r e a t e r t h a n t h a t of n a t i v e t R N A . A t o °, c o n t r a r y to t h e results with phosphodiesterase, c y a n o e t h y l a t e d t R N A
Biochim. Biophys. Acta, 174 (1969) 32-42
38
D. B. MILLAR
0,6
~. 0 . 5 o
ea .
0
0.4
0.3
0.2
0.1
I
I
I
I
I
I
I
I
I
I00
200
300
400
500
600
700
800
900
0.6
~0.s o
0.4 --
20°
m 0 0.3
0.2 0o
O,r I 500
I
tO00
1500 TIME, SECONDS
I 2000
Fig. 4. Ribonuclease (pancreatic) degradation of native t R N A (O) and cyanoethylated t R N A ( 0 ) in o.i M Tris, 0.02 M Mg*+ (pH 8.o) (B); and in 0. 3 M KC1, o.z M Tris (pH 8.0) (A) (2o°). Digestion was started by the addition of 5 o)[ (o. I9 mg/ml) of ribonuclease to 4 ml of a solution of t R N A (o.471 mg/ml).
remains more susceptible to ribonuclease than does native tRNA. Although the over-all rate diminishes from 20 ° to o °, the ratio of ribonuclease liberated acidsoluble nucleotides of cyanoethylated t R N A to that for native tRNA (which I shall call the susceptibility index) remains about the same, approx. 2. In neutral salt, the susceptibility index falls to about 1.5. Several experiments in 0.3 M KC1 studying the rate of ribonuclease digestion as a function of temperature indicated that the difference in susceptibility decreased as the temperature increased. The susceptibility index dropped to about 1.3 at 60 °, the highest temperature studied.
Sephadex chromatography o/ ribonuclease digestion products Fig. 5 shows the separation of ribonuclease digest products of modified t R N A and native tRNA. Digestion was carried on for 225 and IOOO sec for modified and Biochim. Biophys. Acga, 174 (1969) 32-42
TERTIARY STRUCTURE DETERMINANTS IN
tRNA
39
26COO
1,,2o
1,5
I°i°a5 ooo
o
,%
! I [___
^
I0
20
30
410
TUBENUMBER
Fig. 5. G-75 S e p h a d e x c h r o m a t o g r a p h of r i b o n u c l e a s e d i g e s t i o n p r o d u c t s (o.i M Tfis, 0.02 M Mg 2+, p H 8.0, 2o °) of n a t i v e ( G ) a n d c y a n o e t h y l a t e d t R N A ( O ) . C o l u m n d i m e n s i o n s 50.5 c m × i cm. E l u t i n g buffer; o.i M E D T A , p H 7.0. T h e ratio of r i b o n u c l e a s e ( m g / m g ) to t R N A w a s tile s a m e as in Fig. 4- I n all cases, o n l y s l i g h t l y over I m g of d i g e s t w a s p u t o n t h e c o l u m n . F o r o t h e r details, see t e x t .
native tRNA, respectively at 2o ° in the presence of o.o2 M Mg2+. Under these conditions the amount of acid-soluble nucleotides released is identical. The difference in patterns is apparent. This result shows how with equal acid-soluble nucleotides as a criterion of digestion, one can actually have quite different kinds of digestion. At o °, where the susceptibility index shows that cyanoethylated tRNA is still less resistant to ribonuclease attack than native tRNA, the chromatographic patterns showed greater similarity. The native tRNA digest was slightly sharper but otherwise identical to the results obtained at 20 °. For cyanoethylated tRNA there were no easily definable molecular weight zones save that there was a distinguishable peak at the 16 500 molecular weight area. Again using the criterion of equal acid soluble digestion products, digestion in 0.3 M KC1 followed by chromatography on Sephadex G-75 showed little difference in the overall patterns except that there was a leading edge in native tRNA which is absent in modified tRNA. Thus in the absence of magnesium the properties of native and modified tRNA approach each other whether judged by optical, hydrodynamic, or enzymatic criteria. Clearly, however, the enzymatic probes show that the subtle difference in structure between native and modified tRNA is still demonstrable.
Properties o/ribonuclease digests o/cyanoethylated tRNA A final point vital to interpretation of the data presented so far is whether or not the structure assumed by cyanoethylated tRNA is one in which interaction between helical areas is occurring. This point was checked by making a ribonuclease digest such that the resulting solution was comprised of fragments similar to those shown in Fig. 5 and freed of short chain oligonucleotides by ethanolic KC1 (ref. 4). This was checked by direct molecular weight analysis on the digest. The digest's absorbance thermal profiles in 0.3 M KC1 and in 0.02 M Mg2+ were virtually identical. The profile was much less cooperative than that for either intact native or modified tRNA. Viscosity analysis on the digest also showed the importance Biochim. Biophys. Acta, 174 (1969) 32-42
4°
D.B. MILLAR
of the integrity of the molecule. The intrinsic viscosity of the digest in o.3 M KC1, o.oi M Tris (pH 7.0) was 0.07 dl/g while that in 0.02 M Mg~+, o.oi M Tris (pH 7.0) was 0.079 dl/g. These values show that loss of the integral structure of modified tRNA results in a gain in hydrodynamic volume and further that the hydrodynamic compactness usually associated with both native and cyanoethylated tRNA in Mg 2+ is lost for fragmented tRNA. By analogy with similar experiments with native tRNA (ref. 4) the compact conformation of cyanoethylated tRNA is indeed one for which virtually the entire chain length is required. It should be mentioned that the alterations in secondary and tertiary structure reported here are fully compatible with theoretical calculations 24. These authors pointed out that with changes in the ratio of AU to GC base pairs the secondary structure (helicity) should change also.
DISCUSSION
Prior to discussing the data, it may be noted that enzymatic and optical analysis on controls indicated no significant difference between control and native tRNA via methylation for instance. This does not mean that the combination of cyanoethylation and possible methylation might not be important. However, as OFENGAND25 has recently cyanoethylated tRNA without methylation agents present and has observed loss of biological activity we feel the above possibility unlikely. It is pertinent to point out that the observations presented here are made on a chemically modified tRNA which, during the removing of the reactants, passes through stages (dialysis against water, chromatography with water as elutant, and finally lyophilization) in which the maintenance of secondary and tertiary structure is impossible, at least at ordinary temperature a,~e. Consequently, interpretation of the data obtained in structure promoting solvents must properly be performed b y asking the question, "Has the chemical modification prevented or allowed the modified tRNA to form a secondary and tertiary structure identical to native t R N A ? " That it has a tertiary structure of some kind in Mg~+ is shown by the Mg~+ induced stabilization of the absorbance melting curve, protection against ribonuclease and the polarization of fluorescence thermal profile. For native tRNA, these phenomena are characteristic of the assumption of the compact tertiary structure in Mg2+ (ref. 2). It seems reasonable to assume that they indicate the same process in cyanoethylated tRNA. That the tertiary structure and secondary structure is different has been shown by a variety of optical analyses and enzymatic probes in the present report. We suggest that tertiary structure is also involved since it is difficult to see how bases which are located in loops can directly affect the secondary structure of tRNA. If, however, their function is to direct or assist the folding and interaction of helical limbs, the modification of these residues would have important consequences. It is difficult to precisely define the native of the difference but plausible speculations can be given. The phosphodiesterase degradation experiments showed the C-C-A terminus of the molecule to be more accessible to exonucleolytic cleavage than native tRNA. The polarization of fluorescence data, however, showed the terminus not to have gained any freedom of rotation over native tRNA as a result of cyanoethylation which demonstrates no gross disruption in the secondary structure in this area. These data may be reconciled by noting for native tRNA (ref. 27) that the rate of Biochim. Biophys. Acta, 174 (1969) 32-42
TERTIARY STRUCTURE DETERMINANTS IN
tRNA
41
phosphodiesterase digestion is much faster in NaC1 than in Mg 2+. Secondly, the status of the terminus (as regards helical rotational rigidity) for both native t R N A (refs. 2, 16) and cyanoethylated t R N A in KC1 and magnesium at temperatures well below the Tm is very similar. Thus, in cyanoethylated tRNA, the rotationally restraining forces operating on the terminus area are effectively identical to those in native tRNA. Thus it appears that the C-C-A terminus of cyanoethylated t R N A in the presence of Mg z+ is in a state somewhat comparable to that of the terminus of native t R N A in neutral salt. This alteration in environment m a y arise from the loss of a configuration which cannot be formed in cyanoethylated tRNA. For instance, it is possible that the resistance of native tRNA to phosphodiesterase in Mg 2+ (ref. 21) where only the C-C-A terminus is removed at 20 °, m a y be due to the presence of a sterically hindering tertiary structure which exerts its protective effect on the bases just past the C-C-A sequences. If this is so, it would mean that in cyanoethylated tRNA, more of the helical limb terminating in the C-C-A sequences protrudes past the tertiary structure and is more subject to phosphodiesterase degradation. This change in environment could come about by a folding of the helical limbs which is different from that obtaining in native tRNA. Clearly, such an event needs not necessarily disturb intrachain non-covalent internucleotide interaction at the C-C-A terminus. To explain the difference in properties of native and modified t R N A in magnesium, we must assume that there is some specific directive present in native t R N A which operates to form the native tertiary structure which is absent in cyanoethylated tRNA. RAKE AND TENER 10 and OFENGANDz5 have carefully shown that neither solvent effects of the reaction (i.e., methylation by dimethyl sulfoxide) or the small degree of modification of other bases (inosine, thiouridine and uridine) can satisfactorily explain the loss of biological activity and have concluded that modification of pseudouridine is responsible. Thus, the modification of pseudouridine and thiouridine apparently prevent, in part, the formation of a normal secondary and tertiary structure and a relatively subtle different conformation is formed instead. We suggest, therefore, that pseudouridine is a primary determinant in the tertiary structure of tRNA. The manner in which pseudouridine residues can direct the proper folding is difficult to suggest. But in any case, it is clear that other tertiary structure determinants are operative in cyanoethylated tRNA.
REFERENCES i J. R. FRESCO, A. ADAMS, R. ASClONE, D. HENLEY AND T. LINDAHL, Cold Spring Harbor Syrup. Quant. Biol., 21 (1966) 527. 2 n . B. MILLAR AND R. F. STEINER, Biochemistry, 5 (1966) 2289. 3 D. B. MILLAR AND M. MAcKENzIE, Biochemistry, 6 (1967) 2520. 4 D. B. MILLAR AND R. W. BYRNE, Arch. Biochem. Biophys., 119 (1967) 398. 5 J. OFENGAND, Biochem. Biophys. Res. Commun., 18 (1965) 192. 6 R. W. CHAMBERS, Biochemistry, 4 (1965) 219. 7 M. YOSHIDA AND T. UKITA, J. Biochem. Tokyo, 57 (1965) 818. 8 J. OFENGAND, L. CH!d AND H. SCHAEFER, Federation Proc., 25 (1966) 3349. 9 M. YOSHIDA AND T. UKITA, J. Biochem. Tokyo, 58 (I965) 191. IO A. V. RAKE AND G. M. TENER, Biochemistry, 5 (1966) 2992. i i M. YOSHIDA AND T. UKITA, Biochim. Biophys. Acta, 123 (1966) 214. 12 J. OFENGAND, J. Biol. Chem., 242 (1967) 5034. 13 D. A. YI'HANTIS, Biochemistry, 3 (1964) 297.
Biochim. Biophys. Acta, 174 (1969) 32-42
42
n.B.
MILLAR
14 D. B. MILLAR AND R. F. STEINER, Biochim. Biophys. Acta, lO2 (i965) 571. I5 J. R. FRESCO, L. C. KLOTZ AND E. G. RICHARDS, Cold spring Harbor Syrup. Quant. Biol., 28 (I963) 83. 16 M. N. LIPSETT, Biochem. Biophys. Res. Commun., 20 (1965) 224. 17 D. B. MILLAR AND M. MAcKENzIE, Biochem. Biophys. Res. Commun., 23 (1966) 8o 4. 18 M. N. LIPSETT, L. A. HEPPEL AND D. F. BRADLEY, J. Biol. Chem., 236 (1961) 857. 19 A. M. MICHELSON AND C. MONNY, Biochim. Biophys. Acta, 149 (1967) lO 7. 20 D. B. MILLAR, Biochem. Biophys. Res. Commun., 28 (1967) 7 o. 21 G. ZUBAY AND M. TAKAislAMI, Biochem. Biophys. Res. Commun., 15 (1964) 207. 22 S. NISHIMURA AND G. D. NOVELLI, Biochem. Biophys. Res. Commun., 23 (1963) 804. 23 M. LITT AND V. M. INGRAM, Biochemistry, 3 (1964) 56o. 24 B. G. TI/MANYAN AND L. YE. SOTNIKOVA, Biophysics, 12 (1967) I. 25 J. OEENGANO, Federation Proc., 27 (1968) 341. 26 A. TlSSlERES, J. Mol. Biol., I (1959) 36527 T. NIEHI AND G. L. CANTONI, J. Biol. Chem., 238 (1963) 3991.
Biochim. Biophys. Acta, 174 (1968) 32-42
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96064
T E R T I A R Y S T R U C T U R E D E T E R M I N A N T S IN T R A N S F E R RNA I. P S E U D O U R I D I N E "
DAVID B. M I L L A R
Laboratory of Physical Bio6hemistry, Naval Medical Research Institute, National Naval Medical Center, Bethesda, Md. (U.S.A.) (Received J u l y 5th, 1968)
SUMMARY
The structural perturbation effects resulting from the cyanoethylation of Escherichia coli transfer RNA (tRNA) have been studied. The results suggest that: I. The C-C-A terminus area in cyanoethylated tRNA is in a more exposed state than in native tRNA. 2. Cyanoethylated t R N A is more susceptible to nuclease degradation than native. 3. There is a loss of A - U base pairs in cyanoethylated tRNA. 4. Cyanoethylated t R N A is capable of forming a compact tertiary conformation in Mg ~÷ but it is a different one than that obtaining for native tRNA. 5. Cyanoethylated t R N A does not show the same Mg 2+ induced cooperative absorbance melting profile as native tRNA. This is probably due to the altered tertiary conformation.
INTRODUCTION
The concept of tertiary structure in transfer RNA (tRNA) arose because of the observation that during the helix-coil transition of t R N A there were temperature scale divergencies between optical and hydrodynamic data. This appears to be the case in yeast and Escherichia coli t R N A (refs. I, 2) in neutral salt and in E. coli t R N A in the presence of Mg *+ (ref. 2) although for yeast t R N A Mg 2+ it has been suggested 1 that the thermally induced collapse of the secondary and tertiary structure occurs simultaneously. While the secondary structure of E. coli t R N A appears to be generated and largely stabilized by hydrogen-bonding determinants, base stacking and other secondary interactions among the four common bases 3, the determinants of the tertiary conformation are at present unknown. A common feature of the tRNA's whose primary structure is known is that looped-out areas are a direct consequence of arranging the bases into a maximally * F r o m Bureau of Medicine and Surgery, N a v y D e p a r t m e n t , Reseach t a s k MRoo5.o6.ooo4. The opinions in this paper are those of the a u t h o r and do n o t necessarily reflect tke views of t h e N a v y D e p a r t m e n t or the Naval Service at large.
Biochim. Biophys. Acta, i74 (1969) 32-42
TERTIARY STRUCTURE DETERMINANTS IN
tRNA
33
hydrogen-bonded hypothetical secondary structure. Viewed in this manner, the looped-out bases do not contribute directly to the stability of the secondary structure. However, if the helical limbs are folded in a highly restricted manner such that two* or more limbs come into close approximation, interaction between the residues of the looped-out areas becomes possible. The biological expression of tRNA requires a discrete tertiary conformation 1 and such discretion is likely to come from base pairs. If this suggestion is correct, such base pairs become "primary determinants" of tertiary structure and other non-covalent stabilizing interactions become "secondary determinants". As possible candidates for the role of primary determinants there is, a priori, no reason to discard any of the bases in the looped-out areas (saving the anti-codon and bases unable to hydrogen bond). Recent work on the selective modification of pseudouridine residues and accompanying loss of biological activity offers a means to check the possibility of this residue playing a role in the maintenance of the tertiary structure of tRNA. Conditions for the modification of pseudouridine vary considerably 5-8 and solvent conditions play a major role in directing the extent as well as the rate of reaction 9,1°. In our study, the conditions of RAKE AND TENER 1° have been used to cyanoethylate all the pseudouridine residues in tRNA. The choice to cyanoethylate all the residues was made on the basis of the work of YOSHIDAAND U K I T A 9'11 who demonstrated that in the presence of 0. 5 M NaC1 only about 1.2 mole % of the pseudouridine reacted and there were no changes in the melting curves of the modified tRNA.
EXPERIMENTAL
Cyanoethylation of pseudouridine was accomplished by following the procedure described in RAKE AND TENER 1°. Under the solvent conditions chosen (50 % dimethyl sulfoxide, 50 % water, 9 ° min at 37 °) only pseudouridine is the major reactant; less than I mole of uridine/mole t R N A reacts. It was found that rigid precautions against contaminating ribonuclease were necessary. Cyanoethylated t R N A was purified by 2-fold icecold ethanol precipitation (in 0.2 M KC1) and dialysis against water at 4 ° for 18 h. Following this, the cyanoethylated t R N A was placed on a G-75 Sephadex column and eluted with water. The central two-thirds of the peak was taken. It was found that during water elution chromatography of unmodified t R N A on Sephadex various cuts of the eluate did not all display the same degree of aggregation towards Mg 2+ as did bulk tRNA. The aggregation tendency decreased as the cuts progressed from the emerging front to the tailing edge. This m a y indicate that all species of t R N A do not aggregate as readily as others. This finding is under investigation. Cyanoethy]ated t R N A was concentrated by pervaporation, and then lyophilized. The degree of cyanoethylation was followed using the spectrophotometric data of CHAMBERS 6, OFENGAND 12, and the nuclease digestion conditions of * T h e a p p r o x i m a t i o n m u s t be a c c o m p l i s h e d b y m e a n s of specific folding o v e r o t h e r a r e a s of t h e molecule since it h a s b e e n s h o w n for E. coli t R N A (ref. 4) t h a t h i g h m o l e c u l a r w e i g h t f r a g m e n t s or m i x e s of f r a g m e n t s e q u a l l i n g t h e m o l e c u l a r w e i g h t of t R N A do n o t e x h i b i t cooperat i v e b e h a v i o r in Mg ~+, w h i c h is one of t h e d i s t i n g u i s h i n g f e a t u r e s of t h e t e r t i a r y s t r u c t u r e in t h i s solvent. H e n c e , t h e full c h a i n l e n g t h of i n t a c t t R N A is i n v o l v e d in t h e folding n e c e s s a r y to a s s u m e a tertiary conformation.
Biochim. Biophys. Acta, 174 (1969) 32-42
34
D . B . MILLAR
RAKE AND TENER1°. Nucleoside chromatography (two dimensional) was done according to OFENGAND12. Molecular weights were estimated using the YPHANTIS procedure 13. Viscosity measurements were performed as before 8. Coupling of the fluorescent dye, acriflavine, to tRNA was accomplished as outlined by MILLARAND STEINER~. A complete analysis of the apparatus and methods used to measure polarization of fluorescence and the factors affecting the measurements has been published 14. Pancreatic ribonuclease was a product of Sigma Chemical Co. Ribonuclease T-I was a produce of Calbiochem, as was snake venom phosphodiesterase (Russel's viper venom). In testing the action of nucleases on native or modified tRNA, o.3-ml aliquots of the reaction mixture were pipetted into 0.3 ml of icecold 0.25 % uranyl acetate, 4 % perchloric acid*, incubated io min in an ice bath, centrifuged for 5 min at 5 ° at 2500 rev./min in an International PR-I centrifuge; 0.3 ml of the superuatant was added to 0.7 ml of H20 and read at 260 m#. Zero-time blanks were taken. In preparing pancreatic ribonuclease digests for column chromatography, the action of ribonuclease was stopped by the addition of bentonite 4 (suspended in o.I M EDTA). The slurry was incubated at o ° for IO min and the bentonite removed by 20 min centrifugation at 17 ooo rev./min in an International Model B-2o centrifuge at 4 °. I ml of the supernatant was placed on a 50.5 cm × I.O cm column of Sephadex G-75 previously equilibrated with o.I M EDTA. The column was run at room temperature (20-23 °) and o.8-ml fractions were collected. The absorption spectrum (recorded on a Cary Model 14 M) of native and modified tRNA to two different temperatures (IO ° and 80 °) was analyzed by means of the technique of FRESCO, KLOTZ AND RICHARDS15. Absorption melting curves were done as previously described ~.
RESULTS
Number o/pseudouridine residues cyanoethylated It was estimated that 2.3 moles of pseudouridine per mole of tRNA were cyanoethylated after 9 ° rain at 37 ° in 50 % dimethylsulfoxide, 5° % H20, I.O M dimethylaminoethanol, 0.45 M acrylonitrile. E. coli tRNA contains 4-thiouridine is, which as the free base reacts with acrylonitrile 1~. The data indicated that 0. 4 mole per mole tRNA had been modified. This value is lower than that reported by OFENSAND12 and may be due to the alkaline lability of cyanoethylated thiouridine (the pH of the reaction is above II).
Homogeneity, molecular weight, sedimentation coefficient, and viscosity o/cyanoethylated t R N A A plot of In y vs. r ~ for cyanoethylated tRNA was rectilinear and the molecular weight was 25 600. The sedimentation coefficient (ultraviolet optics) of cyanoethylated tRNA was indistinguishable from that of native tRNA. The intrinsic viscosity at 20 ° in o.oi M Tris, 0.02 M Mg 2+ (pH 7.0) was determined to be 0.038 * The author is grateful to Dr. M. SINGER for suggestions concerning the precipitation procedure.
Biockim. Biophys. Acta, 174 (1969) 32-42
TERTIARY STRUCTURE DETERMINANTS IN
tRNA
35
dl/g in fairly good agreement with the value of o.o41 dl/g noted previously*. In o. 3 M KC1, o.oi M "Iris (pH 7.o), the value is 0.054 dl/g in good agreement with the value of 0.059 reported by MILLARAND MACKENZIE17. This data indicates that no gross hydrodynamic changes occurred in tRNA upon cyanoethylation and that like native tRNA, cyanoethylated tRNA is able to assume a compact configuration in the presence of Mg*+.
A bsorbance melting curves
Fig. I shows the absorbance melting curves of cyanoethylated tRNA compared to native tRNA in neutral salt and in Mgz+. In agreement with the results of RAKE AND TENERTM there is virtually no difference in thermal denaturation behavior between native and modified tRNA in neutral salt although the maximum absorbance increase is slightly less. In Mgz+, however, the melting profile of modified tRNA is less sharp and cooperative. Controls (omission of acrylonitrile in the incubation reaction) showed only a slight difference from native tRNA melting curves in magnesium.
1.30
t.25
1.20
1,15
P.IO
1.05
0
B d
1.3o
1,25
1.20
I, i5
i. lO
LOS
I 0
I0
r~
t~ 20
I 30
40
50
60
70
80
90
TEMPERATURE
Fig. I. Melting profiles of n a t i v e (C)) a n d c y a n o e t h y l a t e d t R N A ( O ) . (A) 0.02 M Mg 2+, o.oi M Tris (pH 7.o); (B) 0. 3 M KC1, o.oi M Tris (pH 7.0). I n b o t h c a s e s t h e u p p e r s e t of p o i n t s a t 20 ° i n d i c a t e s t h e reversible n a t u r e of tile profile.
Biochim. Biophys. Acta, 174 (1969) 32-42
36
D.B. MILLAR
Analysis o/ the thermal denaturation spectrum The absorbance melting profiles were analyzed by the method of FRESCO, KLOTZ AND RICHARDS15. There m a y be slightly more GC pairs melting out in native TABLE I BASE PAIRS MELTING OUT BETWEEN VARIOUS TEMPERATURES
Temperature range (°C)
Soh, ent
IO-80 lO-61 lO-8O
o.o2 M M g * + + o . o i M Tris (pH 7.o) o.o2 M M g * + + o . o I M Tris (pH 7.0) 0. 3 M K C l + o . o i M Tris (pH 7.0)
Native t R N A
Cyanoethylated t R N A
IAu*
IGC*
IAU*
/GC**
o.31
o.47
0.32
0.46
o.24 ,~o.o8 0.24
o.43 ,-~o.o8 0.45
* fAU and fGC axe the fractions of total nucleotides in AU or GC pairs, respectively.
than in cyanoethylated tRNA and there is approximately a 2o % decrease in AU pairs melted out in cyanoethylated t R N A as compared with native. In view of the high temperature, the plateauing of the absorbance curve of cyanoethylated t R N A (Fig. I) and the known temperature stability of short length AU pairs 18,x9 a reasonable interpretation of this result would be that there are less AU pairs in cyanoethylated t R N A than in native tRNA. The temperature zone between io ° and 61 ° in magnesium was also investigated since it is here that differences in the melting curve of modified t R N A vs. native t R N A are first seen. It should be pointed out that the values of fAU and fac estimated for this temperature range are very imprecise (4-20 %) due to the small amplitude of the denaturation spectra (Table I). The values indicate that there are a small number of AU and GC pairs which are noncooperatively melting out before the remainder of the AU and GC pairs in cyanoethylated tRNA.
Effect o/cyanoethylation on helical rigidity The question arises as to what effect the modification of t R N A has on helical rigidity; that is, the rotational freedom of the bases. This point was investigated by means of the experiment shown in Fig. 2 which shows a plot of I / P + I / 3 vs. Tin. Here P is a polarization of fluorescence, T is absolute temperature, and n is solvent viscosity. In this experiment, loss of internal rotational rigidity which accompanies the disruption of the ordered structure of a polynucleotide ~'14 is signaled by a drop of P, hence I / P + I / 3 rises. Comparison of this data with that for native t R N A (refs. 2, I7) shows that the thermally induced acquisition of rotational freedom is virtually the same in cyanoethylated tRNA as it is in native tRNA. This result implies that neither the base pairs melting out early nor the "lost" AU pairs in cyanoethylated t R N A (Table I) cannot all be concentrated adjacent to the C-C-A terminus where the fluorescent label is bound (on the ribose of the adenosine residue). This follows since if all the base pairs were in this area, their thermal dissociation (and increase in rotational freedom) should have been reflected by a decrease in P markedly different from native t R N A (ref. 20). Such is not the case. If, however, Bioehim. Biophys. Acta, 174 (1969) 32-42
TERTIARY STRUCTURE DETERMINANTS IN t R N A
.o}
37
TEMPERATI.RZ 75
2'
~25
42
5:
5~
6~
0.~
5O
0 I
4oh g
30) ~o~ s'
°[
,;o ~'o ~o
o'o ~'0 ,;0
,~
go
tOO
ZSO
30~
._ •
40c
:JME, M~NUTES
Fig. 2. Polarization of fluorescence thermal ~ofile of an acriflavine conjugate of cyanoethylated tRNA. O, 0.3 M KC1, o.oi M Tris (pH 7.0); 0 , 0.02 MMg *+, o.oi MTris (pH 7.o). The temperature scale is adjusted to be in register with values of T/n. Concentration approx. 0. 3 mg/ml. ~ excitation 460 m/~; emission 5IO m/z. Fig. 3. Phosphodiesterase digestion of native tRNA (O) and cyanoethylated tRNA (O). Solvent: o.i M Tris, 0.02 M Mg*+ (pH 8.o). Digestion was started by the addition of i ml of phosphodiesterase (i5o units/ml in tile above solvent) to 2 ml of a tRNA solution; final concentratiort of tRNA: o.314 mg/ml. The extent of digestion was determined as described in the text.
the bases were localized n e a r the C - C - A t e r m i n u s a n d it was i m m o b i l i z e d b y some o t h e r r o t a t i o n a l l y r e s t r a i n i n g forces (e.g., either efficient base s t a c k i n g or the i n t i m a t e p r o x i m i t y of o t h e r helical regions - - i.e., " t i g h t l y b u r i e d " within the molecule) t h e n t h e s i t u a t i o n m i g h t possibly be rationalized.
Action o/venom phosphodiesterase on modified t R N A To check t h e l a t t e r point, t h e action of v e n o m p h o s p h o d i e s t e r a s e on n a t i v e a n d c y a n o e t h y l a t e d t R N A was investigated. W i t h b o t h phosphodiesterase a n d ribonuclease digestion (reported on below), controls were g e n e r a l l y digested at i d e n t i c a l rates as n a t i v e t R N A ; a l t h o u g h in some cases there was a slight increase (approx. IO % ) in rates of digestion. I t is seen (Fig. 3) t h a t a t 37 °, t h e action of p h o s p h o d i e s t e r ase is twice as extensive on c y a n o e t h y l a t e d t R N A as on native. This indicates t h a t t h e nuclease a t t a c k proceeds p a s t t h e C - C - A m o i e t y in modified t R N A ; since in n a t i v e t R N A , nuclease a t t a c k ceases a t a b o u t this p o i n t zl (in Mg 2+ at 20°). This d a t a indicates t h e C - C - A t e r m i n u s a n d t h e i m m e d i a t e base pairs n e x t to it are not d e e p l y a n d / o r rigidly b u r i e d in t h e s t r u c t u r e of c y a n o e t h y l a t e d t R N A . A t o °, n a t i v e a n d c y a n o e t h y l a t e d t R N A are e q u a l l y a t t a c k e d b y phosphodiesterase, i n d i c a t i n g t h a t b e t w e e n 37 ° a n d o ° some e v e n t has occurred in c y a n o e t h y l a t e d t R N A which confers p r o t e c t i o n a g a i n s t t h e nuclease.
Action o/ribonuclease on cyanoethylated t R N A N a t i v e t R N A in a similar Mg 2+ c o n t a i n i n g solvent is r e l a t i v e l y r e s i s t a n t t o the action of ribonuclease 4,23.~4. A t 20 ° (Fig. 4) with c y a n o e t h y l a t e d t R N A b o t h t h e initial r a t e a n d e x t e n t of d e g r a d a t i o n are c o n s i d e r a b l y g r e a t e r t h a n t h a t of n a t i v e t R N A . A t o °, c o n t r a r y to t h e results with phosphodiesterase, c y a n o e t h y l a t e d t R N A
Biochim. Biophys. Acta, 174 (1969) 32-42
38
D. B. MILLAR
0,6
~. 0 . 5 o
ea .
0
0.4
0.3
0.2
0.1
I
I
I
I
I
I
I
I
I
I00
200
300
400
500
600
700
800
900
0.6
~0.s o
0.4 --
20°
m 0 0.3
0.2 0o
O,r I 500
I
tO00
1500 TIME, SECONDS
I 2000
Fig. 4. Ribonuclease (pancreatic) degradation of native t R N A (O) and cyanoethylated t R N A ( 0 ) in o.i M Tris, 0.02 M Mg*+ (pH 8.o) (B); and in 0. 3 M KC1, o.z M Tris (pH 8.0) (A) (2o°). Digestion was started by the addition of 5 o)[ (o. I9 mg/ml) of ribonuclease to 4 ml of a solution of t R N A (o.471 mg/ml).
remains more susceptible to ribonuclease than does native tRNA. Although the over-all rate diminishes from 20 ° to o °, the ratio of ribonuclease liberated acidsoluble nucleotides of cyanoethylated t R N A to that for native tRNA (which I shall call the susceptibility index) remains about the same, approx. 2. In neutral salt, the susceptibility index falls to about 1.5. Several experiments in 0.3 M KC1 studying the rate of ribonuclease digestion as a function of temperature indicated that the difference in susceptibility decreased as the temperature increased. The susceptibility index dropped to about 1.3 at 60 °, the highest temperature studied.
Sephadex chromatography o/ ribonuclease digestion products Fig. 5 shows the separation of ribonuclease digest products of modified t R N A and native tRNA. Digestion was carried on for 225 and IOOO sec for modified and Biochim. Biophys. Acga, 174 (1969) 32-42
TERTIARY STRUCTURE DETERMINANTS IN
tRNA
39
26COO
1,,2o
1,5
I°i°a5 ooo
o
,%
! I [___
^
I0
20
30
410
TUBENUMBER
Fig. 5. G-75 S e p h a d e x c h r o m a t o g r a p h of r i b o n u c l e a s e d i g e s t i o n p r o d u c t s (o.i M Tfis, 0.02 M Mg 2+, p H 8.0, 2o °) of n a t i v e ( G ) a n d c y a n o e t h y l a t e d t R N A ( O ) . C o l u m n d i m e n s i o n s 50.5 c m × i cm. E l u t i n g buffer; o.i M E D T A , p H 7.0. T h e ratio of r i b o n u c l e a s e ( m g / m g ) to t R N A w a s tile s a m e as in Fig. 4- I n all cases, o n l y s l i g h t l y over I m g of d i g e s t w a s p u t o n t h e c o l u m n . F o r o t h e r details, see t e x t .
native tRNA, respectively at 2o ° in the presence of o.o2 M Mg2+. Under these conditions the amount of acid-soluble nucleotides released is identical. The difference in patterns is apparent. This result shows how with equal acid-soluble nucleotides as a criterion of digestion, one can actually have quite different kinds of digestion. At o °, where the susceptibility index shows that cyanoethylated tRNA is still less resistant to ribonuclease attack than native tRNA, the chromatographic patterns showed greater similarity. The native tRNA digest was slightly sharper but otherwise identical to the results obtained at 20 °. For cyanoethylated tRNA there were no easily definable molecular weight zones save that there was a distinguishable peak at the 16 500 molecular weight area. Again using the criterion of equal acid soluble digestion products, digestion in 0.3 M KC1 followed by chromatography on Sephadex G-75 showed little difference in the overall patterns except that there was a leading edge in native tRNA which is absent in modified tRNA. Thus in the absence of magnesium the properties of native and modified tRNA approach each other whether judged by optical, hydrodynamic, or enzymatic criteria. Clearly, however, the enzymatic probes show that the subtle difference in structure between native and modified tRNA is still demonstrable.
Properties o/ribonuclease digests o/cyanoethylated tRNA A final point vital to interpretation of the data presented so far is whether or not the structure assumed by cyanoethylated tRNA is one in which interaction between helical areas is occurring. This point was checked by making a ribonuclease digest such that the resulting solution was comprised of fragments similar to those shown in Fig. 5 and freed of short chain oligonucleotides by ethanolic KC1 (ref. 4). This was checked by direct molecular weight analysis on the digest. The digest's absorbance thermal profiles in 0.3 M KC1 and in 0.02 M Mg2+ were virtually identical. The profile was much less cooperative than that for either intact native or modified tRNA. Viscosity analysis on the digest also showed the importance Biochim. Biophys. Acta, 174 (1969) 32-42
4°
D.B. MILLAR
of the integrity of the molecule. The intrinsic viscosity of the digest in o.3 M KC1, o.oi M Tris (pH 7.0) was 0.07 dl/g while that in 0.02 M Mg~+, o.oi M Tris (pH 7.0) was 0.079 dl/g. These values show that loss of the integral structure of modified tRNA results in a gain in hydrodynamic volume and further that the hydrodynamic compactness usually associated with both native and cyanoethylated tRNA in Mg 2+ is lost for fragmented tRNA. By analogy with similar experiments with native tRNA (ref. 4) the compact conformation of cyanoethylated tRNA is indeed one for which virtually the entire chain length is required. It should be mentioned that the alterations in secondary and tertiary structure reported here are fully compatible with theoretical calculations 24. These authors pointed out that with changes in the ratio of AU to GC base pairs the secondary structure (helicity) should change also.
DISCUSSION
Prior to discussing the data, it may be noted that enzymatic and optical analysis on controls indicated no significant difference between control and native tRNA via methylation for instance. This does not mean that the combination of cyanoethylation and possible methylation might not be important. However, as OFENGAND25 has recently cyanoethylated tRNA without methylation agents present and has observed loss of biological activity we feel the above possibility unlikely. It is pertinent to point out that the observations presented here are made on a chemically modified tRNA which, during the removing of the reactants, passes through stages (dialysis against water, chromatography with water as elutant, and finally lyophilization) in which the maintenance of secondary and tertiary structure is impossible, at least at ordinary temperature a,~e. Consequently, interpretation of the data obtained in structure promoting solvents must properly be performed b y asking the question, "Has the chemical modification prevented or allowed the modified tRNA to form a secondary and tertiary structure identical to native t R N A ? " That it has a tertiary structure of some kind in Mg~+ is shown by the Mg~+ induced stabilization of the absorbance melting curve, protection against ribonuclease and the polarization of fluorescence thermal profile. For native tRNA, these phenomena are characteristic of the assumption of the compact tertiary structure in Mg2+ (ref. 2). It seems reasonable to assume that they indicate the same process in cyanoethylated tRNA. That the tertiary structure and secondary structure is different has been shown by a variety of optical analyses and enzymatic probes in the present report. We suggest that tertiary structure is also involved since it is difficult to see how bases which are located in loops can directly affect the secondary structure of tRNA. If, however, their function is to direct or assist the folding and interaction of helical limbs, the modification of these residues would have important consequences. It is difficult to precisely define the native of the difference but plausible speculations can be given. The phosphodiesterase degradation experiments showed the C-C-A terminus of the molecule to be more accessible to exonucleolytic cleavage than native tRNA. The polarization of fluorescence data, however, showed the terminus not to have gained any freedom of rotation over native tRNA as a result of cyanoethylation which demonstrates no gross disruption in the secondary structure in this area. These data may be reconciled by noting for native tRNA (ref. 27) that the rate of Biochim. Biophys. Acta, 174 (1969) 32-42
TERTIARY STRUCTURE DETERMINANTS IN
tRNA
41
phosphodiesterase digestion is much faster in NaC1 than in Mg 2+. Secondly, the status of the terminus (as regards helical rotational rigidity) for both native t R N A (refs. 2, 16) and cyanoethylated t R N A in KC1 and magnesium at temperatures well below the Tm is very similar. Thus, in cyanoethylated tRNA, the rotationally restraining forces operating on the terminus area are effectively identical to those in native tRNA. Thus it appears that the C-C-A terminus of cyanoethylated t R N A in the presence of Mg z+ is in a state somewhat comparable to that of the terminus of native t R N A in neutral salt. This alteration in environment m a y arise from the loss of a configuration which cannot be formed in cyanoethylated tRNA. For instance, it is possible that the resistance of native tRNA to phosphodiesterase in Mg 2+ (ref. 21) where only the C-C-A terminus is removed at 20 °, m a y be due to the presence of a sterically hindering tertiary structure which exerts its protective effect on the bases just past the C-C-A sequences. If this is so, it would mean that in cyanoethylated tRNA, more of the helical limb terminating in the C-C-A sequences protrudes past the tertiary structure and is more subject to phosphodiesterase degradation. This change in environment could come about by a folding of the helical limbs which is different from that obtaining in native tRNA. Clearly, such an event needs not necessarily disturb intrachain non-covalent internucleotide interaction at the C-C-A terminus. To explain the difference in properties of native and modified t R N A in magnesium, we must assume that there is some specific directive present in native t R N A which operates to form the native tertiary structure which is absent in cyanoethylated tRNA. RAKE AND TENER 10 and OFENGANDz5 have carefully shown that neither solvent effects of the reaction (i.e., methylation by dimethyl sulfoxide) or the small degree of modification of other bases (inosine, thiouridine and uridine) can satisfactorily explain the loss of biological activity and have concluded that modification of pseudouridine is responsible. Thus, the modification of pseudouridine and thiouridine apparently prevent, in part, the formation of a normal secondary and tertiary structure and a relatively subtle different conformation is formed instead. We suggest, therefore, that pseudouridine is a primary determinant in the tertiary structure of tRNA. The manner in which pseudouridine residues can direct the proper folding is difficult to suggest. But in any case, it is clear that other tertiary structure determinants are operative in cyanoethylated tRNA.
REFERENCES i J. R. FRESCO, A. ADAMS, R. ASClONE, D. HENLEY AND T. LINDAHL, Cold Spring Harbor Syrup. Quant. Biol., 21 (1966) 527. 2 n . B. MILLAR AND R. F. STEINER, Biochemistry, 5 (1966) 2289. 3 D. B. MILLAR AND M. MAcKENzIE, Biochemistry, 6 (1967) 2520. 4 D. B. MILLAR AND R. W. BYRNE, Arch. Biochem. Biophys., 119 (1967) 398. 5 J. OFENGAND, Biochem. Biophys. Res. Commun., 18 (1965) 192. 6 R. W. CHAMBERS, Biochemistry, 4 (1965) 219. 7 M. YOSHIDA AND T. UKITA, J. Biochem. Tokyo, 57 (1965) 818. 8 J. OFENGAND, L. CH!d AND H. SCHAEFER, Federation Proc., 25 (1966) 3349. 9 M. YOSHIDA AND T. UKITA, J. Biochem. Tokyo, 58 (I965) 191. IO A. V. RAKE AND G. M. TENER, Biochemistry, 5 (1966) 2992. i i M. YOSHIDA AND T. UKITA, Biochim. Biophys. Acta, 123 (1966) 214. 12 J. OFENGAND, J. Biol. Chem., 242 (1967) 5034. 13 D. A. YI'HANTIS, Biochemistry, 3 (1964) 297.
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n.B.
MILLAR
14 D. B. MILLAR AND R. F. STEINER, Biochim. Biophys. Acta, lO2 (i965) 571. I5 J. R. FRESCO, L. C. KLOTZ AND E. G. RICHARDS, Cold spring Harbor Syrup. Quant. Biol., 28 (I963) 83. 16 M. N. LIPSETT, Biochem. Biophys. Res. Commun., 20 (1965) 224. 17 D. B. MILLAR AND M. MAcKENzIE, Biochem. Biophys. Res. Commun., 23 (1966) 8o 4. 18 M. N. LIPSETT, L. A. HEPPEL AND D. F. BRADLEY, J. Biol. Chem., 236 (1961) 857. 19 A. M. MICHELSON AND C. MONNY, Biochim. Biophys. Acta, 149 (1967) lO 7. 20 D. B. MILLAR, Biochem. Biophys. Res. Commun., 28 (1967) 7 o. 21 G. ZUBAY AND M. TAKAislAMI, Biochem. Biophys. Res. Commun., 15 (1964) 207. 22 S. NISHIMURA AND G. D. NOVELLI, Biochem. Biophys. Res. Commun., 23 (1963) 804. 23 M. LITT AND V. M. INGRAM, Biochemistry, 3 (1964) 56o. 24 B. G. TI/MANYAN AND L. YE. SOTNIKOVA, Biophysics, 12 (1967) I. 25 J. OEENGANO, Federation Proc., 27 (1968) 341. 26 A. TlSSlERES, J. Mol. Biol., I (1959) 36527 T. NIEHI AND G. L. CANTONI, J. Biol. Chem., 238 (1963) 3991.
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