10
Biochimica et Biophysica Acta, 477 ( 1 9 7 7 ) 1 0 - - 1 9 © Elsevier/North-Holland Biomedical
Press
BBA 9 8 9 4 4
E F F E C T O F MAGNESIUM AND POLYAMINES ON THE S T R U C T U R E O F YEAST t R N A Phe
P.H. B O L T O N a n d D.R. K E A R N S
Department of Chemistry, University of California, San Diego, La Jolla, Calif. 92093 (U.S.A.) ( R e c e i v e d N o v e m b e r 23rd, 1976)
Summary The effect of magnesium and polyamines (spermine, spermidine, putrescine and cadaverine) on the structure of yeast t R N A Phe has been investigated. It is found that magnesium induces structural changes and stabilizes hydrogen bonds in the temperature range 22--44°C in 0.17 M sodium. The number of Mg 2÷ which affect t R N A structure increases from 1 -+ 1 at 22°C to 4 + 1 at 44°C and the number of additional base pairs formed in the presence of magnesium increases from 1 + 1 at 22°C to 4 + 1 at 44°C. The spectral changes are more-or-less sequential. The polyamine spermine stabilizes some, b u t not all, of the structural features stabilized b y magnesium at 44 ° C, and the combination of magnesium and spermine, at low levels, is more effective than either cation alone in stabilizing t R N A structure. Comparison of the effects of spermine, spermidine, putrescine and cadaverine indicates that it is the asymmetric triamine unit which is important in the stabilization. Some spectral changes induced by magnesium can be assigned to stabilization of specific tertiary structure interactions and to alteration of stacking adjacent to U8 • A~4.
Introduction It has been recognized for some time that polyvalent cations in general and magnesium in particular are important for the biological function and native structure of t R N A [1--19]. Previous studies on the interaction of polyvalent cations with t R N A have a t t e m p t e d to determine the strength and number of binding sites, as well as the effect of polyvalent cations on t R N A structure. A n u m b e r of reports have appeared which conclude that t R N A have a b o u t five A b b r e v i a t i o n s : p p m , parts p e r million; EDTA, e t h y l e n e d i a m i n e t e t r a a c e t i c acid; a m o u n t o f m a t e r i a l t h a t h a d a n a b s o r b a n c e o f 1 . 0 at 2 6 0 n m w h e n d i s s o l v e d in m e a s u r e d in a 1 c m P a t h .
A260 unit, an 1 ml w a t e r a n d
11 strong binding sites for magnesium, K ~ 10SM -1, in a wide variety of experimental conditions [1,5,11--13,15,16]. However, a recent report by Stein and Crothers [2--4] indicates that the number of binding sites, as well as the strength of binding, depend on the concentration of Na ÷. Differential melting [1], tritium exchange [10], temperature jump [3,4,14,17] and Raman spectroscopy [8,9] indicate that magnesium has a pronounced effect on tRNA structure in the presence of high levels of sodium at low temperature while the results of some circular dichroism [10], tritium exchange [10] and temperature jump studies [3,4,14,17] indicate that magnesium has little effect on tRNA structure in these conditions. Similarly, the results of previous NMR investigations are in disagreement as to whether magnesium affects the structure of tRNA at low temperature in the presence of high levels of sodium [6,7,20-23]. While these earlier investigations have answered certain questions regarding the general nature of the tRNA-magnesium interaction, important details remain to be elucidated. For example: which structural features are affected by magnesium, and in which order are they affected? Is tRNA structure the same in the presence of sodium as in the presence of magnesium? To what extent do the details of the tRNA-magnesium interaction depend on experimental conditions, i.e. salt and temperature? NMR is particularly well suited to the investigation of the effect of magnesium on tRNA structure since two kinds of effects are observable, stabilization of base pairs or other hydrogen bonding interactions and structural change [6,7,24,25]. The life-time of a proton in a hydrogen-bonded state (as in a base pair) may be too short to be observed by NMR in the absence of magnesium, but in the presence of magnesium, the life-time may be increased to the point where it becomes observable. This effect, which we shall refer to as "stabilization" of a hydrogen bonding interaction, is manifested as an increase in the intensity of the low field region of the NMR spectrum. Magnesium may induce a change in the conformation of the tRNA and this will be manifested in the shift of resonances which were already present in the spectrum in the absence of magnesium. Both kinds of effects are observed in the interaction of magnesium with tRNA. In the present investigation, we have examined the effect of magnesium on the NMR spectrum of yeast tRNA Phe at several different temperatures ranging from 22--44°C. When excess magnesium is present, the tRNA exhibit 3--4 more resonances in the low field NMR spectrum than would be expected from the cloverleaf secondary structure base pairs alone [6,7,25]. These extra resonances have been assigned to specific tertiary interactions [6,7,21,22,25--27] and thus NMR allows both the secondary and tertiary structural features in the tRNA to be monitored. By comparing spectra of yeast tRNA Phe obtained in the presence of varying amounts of magnesium with the spectrum obtained in the presence of excess magnesium (taken to be the reference state, presumably native structure), we are able to determine the number of base pairs stabilized, the number of Mg2÷ which are effective in stabilizing the tertiary structure, and the order in which structural features are stabilized. Some information about the structural rearrangements which are induced by magnesium is also obtained.
12 The affect of polyamines on t R N A structure was also examined since certain polyamines can replace magnesium, at least partially, in stimulating the biological activity of t R N A in vitro [5,9]. The polyamine-tRNA interaction is also of interest since polyamines have been used in growing crystals of t R N A used in the X-ray diffraction studies [28,29]. Materials and Methods Unfractionated yeast t R N A was obtained from Plenum Scientific. Yeast t R N A Phe was purified to a phenylalanine acceptance of at least 1.5 nM/A260 by use of BD-cellulose [30], RPC-5 [31] and RPC-7 [32] chromatography. The amino acyl ligase for the assay of t R N A was prepared as described elsewhere [33]. After purification the t R N A samples were extensively dialysed against 0.5 M NaC1 and 5 mM EDTA at pH 7 to remove magnesium. The t R N A samples were then vacuum dialysed against the buffer used in the NMR experiments: 0.1 M NaC1 and 10 mM K2H2PO4 at pH 7. The concentration of tRNA was I mM so the total concentration of sodium was about 0.17 M (0.1 M free a n d about 0.07 M b o u n d to tRNA). A sodium concentration of 0.17 M was chosen to minimize the changes in ionic strength during the cation titrations and to allow comparison of these results with those obtained previously [2,3, 10,14]. Furthermore, in the presence of 0.17 M sodium at 22°C, t R N A has a compact folded structure similar to the native structure [2,3,10,14]. The tRNA concentration was determined by assuming that 1.6 nM of t R N A dissolved in 1 ml of distilled w a t e r has an absorbance of 1.0 at 260 nm. The magnesium and polyamine titrations were performed by adding the proper a m o u n t o f 10 mM cation to the NMR sample followed by an equimolar a m o u n t ' o f K2H2PO4 at pH 7 to ensure a constant pH. The sample was then reduced to its original volume by a stream of dry, filtered nitrogen. In this manner, reproducible spectral intensities of better than 5% were obtained. The NMR spectra were obtained on a Varian HR-300 operated in the continuous wave field sweep mode. The spectra were signal averaged 1--2 h on a Nicolet 1020A to improve the signal-to-noise ratio. Results and Discussion
Effect of magnesium on tRNA structure T h e spectra in Fig. 1 show t h e effect of magnesium on the low field spect r u m on yeast t R N A ehe at 22°C. The addition of 1 Mg2+/tRNA does n o t increase the intensity of the low field region indicating there is no net gain in the n u m b e r of base pairs. However, a resonance initially located at 14.1 ppm in the absence of magnesium shifts to 14.4 ppm. In other regions of the spectrum t h e r e are smaller shifts and gains of intensity. The addition of a second magnesium per t R N A induces a gain of intensity near 13.2 ppm, as well as several small shifts of intensity t h r o u g h o u t the spectrum. The addition of more than 2 Mg2+/tRNA has little, or no, effect on the spectrum. Thus, at 22°C in the presence of 0.17 M Na ÷, magnesium induces the gain of intensity of o n l y 1 + 1 resonance, and the spectral changes which do occur are sequential. For example, the first magnesium strongly affects the spectral region between 14.1 and 14.4 ppm and induces little gain of intensity near 13.2 ppm, whereas the second
13 A Mg/tRN~,
-
2
[ ,5 (o)
//
yeost tRNA Phe T=22°
'1
L r4
I
,13 ppm
15 (b)
I
14
I
13
I
12
I
II
ppm
Fig. I . E f f e c t o n m a g n e s i u m o n t h e 3 0 0 M H z N M R s p e c t r u m o f yeast t R N A Phe at 22°C. To en~phasize the spectral changes, the spectra at different l e v e l s o f m a g n e s i u m a r e drawn superimposed. The darkened a r e a s r e p r e s e n t r e g i o n s w h e r e t h e r e is a g a i n o f intensity w h e n the m a g n e s i u m c o n c e n t r a t i o n is i n c r e a s e d . The shaded areas indicate regions where there is a loss of intensity w h e n the magnesium c o n c e n t r a t i o n is increased.
magnesium has little affect on the spectral region between 14.1 and 14.4 ppm but it does induce a gain of intensity near 13.2 ppm. The shift of the resonance position of Us • A14 might be due to movement of Gls since the crystal structure o f yeast tRNA Phe predicts that this residue exerts the largest ring current shift on U8 • A14 [35--38]. The stabilization of the G~9 • Cs6 tertiary base pair indicates t h a t this is the weakest tertiary interaction in the molecule we are able to monitor. The spectra in Fig. 2 show the effect of magnesium on the low field spectrum of yeast tRNA Ph" at 37°C. The addition of 1 Mg2+/tRNA induces a shift of intensity from 14.1 to 14.4 ppm as was observed at 22°C and a gain of intensity at 13.2 ppm. The further addition of magnesium to 2 / t R N A induces a gain o f intensity at 13.2 and 12.4 ppm. The spectral changes, again, are moreor-less sequential. The addition of more than 2 Mg2÷/tRNA does not increase the intensity o f the spectrum but it does induce small shifts in the positions of several resonances. The t o t a l gain in intensity induced by magnesium corresponds to 2 -+ 1 base pairs, in agreement with the number reported previously [6,7]. The spectra in Fig. 3 show the effect of magnesium on the low field spectrum of yeast t R N A phe at 44°C. The addition of 2 Mg2+/tRNA induces major gains of intensity at 14.4, 13.8 and 11.5 ppm, as well as loss of intensity at 13.6 and 12.8 ppm. Further addition of magnesium to 4 / t R N A induces some
14 yeast tRNAPhe T=37 ° Mg/tRNA
.
~
.
GAIN IN INTENSITY WITH Mg (e) LO S S (~)
yeast tRNA Phe T=37 °
2vsl M p ~ ~
IvsO M
I
I
15 (o)
14
I
13
1
I
12
pp m
II
.....
(b)
~
I
I
15
I
14
I
13 ppm 12
F i g . 2. E f f e c t o f m a g n e s i u m o n t h e 3 0 0 M H z N M R s p e c t r u m o f y e a s t t R N A P h e at 3 7 ° C . See Fig. 1 f o r additional details.
',, yeast tRNAPhe '! T =4 4 °
Mg
VI'i
GAIN IN INTENSITY WITH Mg (o)
/!\
yeast tRNA Phe T=44°
LOSS (~,)
' I~1
.i ((3)
I5
/~v
1
i
i
I
i
~4
13
12
n
Io
ppm
I
15 (13)
I
14
I
13
_
J
12
ppm
F i g . 3. E f f e c t o f m a g n e s i u m on t h e 3 0 0 M H z N M R s p e c t r u m o f y e a s t t R N A P h e at 4 4 ° C . See F i g . 1 f o r additional details.
15 additional gains in intensity, b u t above 4 Mg~÷/tRNA there is no increase in intensity in the low field region of the spectrum, although there are small shifts in the positions of several resonances. Comparison of the spectral changes induced by magnesium shows that the stabilization is more-or-less sequential and the total number o f additional base pairs stabilized by magnesium is 4 + 1; this number also agrees with the number previously reported [34]. The results presented here on yeast t R N A Phe indicate that at 44°C, there is the gain of intensity at 14.4, 13.8, 13.2 and 11.5 ppm (as well as at other positions), and these are positions at which the resonances of the tertiary interactions U8 A~4, mlAss • T54, G19 • Cs6, and U33 • P36 have been assigned [7,25,34]. Thus it appears that magnesium stabilizes the tertiary structure of yeast t R N A Phe at 44°C as well as certain features of the secondary structure. At 37°C the tertiary structure is only partially destabilized and, hence, magnesium only stabilizes G19 • Cs6 and affects the stacking of residues adjacent to Us • A14.
Effect o f polyamines on tRNA structure The spectra in Fig. 4 show the effect of the polyamine spermine on the low field spectrum of yeast t R N A Phe at 44°C. Addition of 1 spermine/tRNA induces gain of intensity, primarily at 13.8 ppm, and further addition of spermine to 3 / t R N A induces gain of intensity near 14.4, 13.2, 12.4, as well as at 13.8 ppm. Above 3 spermine/tRNA, there is little or no effect on the spectrum b u t at 1 0 / t R N A the t R N A begins to precipitate. Thus, the spectra indicate that a b o u t 3 spermine/tRNA stabilize certain features of t R N A structure and that the stabilization is more-or-less sequential. The total n u m b e r of base pairs stabilized by spermine is 2 + 1, which is less than the total number stabilized by magnesium under otherwise identical conditions.
S, erm,n,,It' 'A GAIN IN INTENSITY WITH SPERMINE (e)
yeast tRNA Phe T=44°
LOSS (,-)
vs0y .l
16 (al
I
15
r
14
I
13
I
pprn
12
I
fl
I
IO
I
9
I
(b)
15
I
14
l
13 ppm
I
12
Fig. 4. E f f e c t o f s p e r m i n e o n t h e 3 0 0 M H z N M R s p e c t r u m o f y e a s t t R N A Phe at 4 4 ° C . See Fig. 1 f o r a d d i t i o n a l details.
16 The spectra in Fig. 5 show the combined effect of spermine and magnesium on the low field spectrum of yeast t R N A Phe at 44°C. The addition of 1 Mg2+/ t R N A to t R N A in the presence of 3 spermine/tRNA and 0.17 M Na* induces gains of intensity mainly at 14.4, 13.2 and 12.4 ppm. The further addition of magnesium to 2 / t R N A induces gain of intensity primarily near 12.4 and 13.3 ppm; the spectral changes are seen to be more-or-less sequential. Addition of more than 2 Mg2÷/tRNA produces no further increase in the intensity of the low field region, although there are several small shifts of intensity such t h a t at 10 Mg2÷/tRNA the spectrum is essentially identical to that of tRNA in the presence of 10 Mg2÷/tRNA and no spermine. The addition of magnesium to t R N A in the presence of 3 spermine/tRNA induces the gain of intensity of 2 + 1 resonances. The polyamine spermidine was f o u n d to affect the spectrum of t R N A in a manner nearly identical to t h a t of spermidine, but the diamines putrescine and cadaverine were f o u n d to have little or no effect on t R N A spectra, even at 1 0 / t R N A at 44°C. This indicates that it is the asymmetric triamine unit which is important in the stabilization.
:
)!I
Mg/tRNA
yeast
,~, I'
~RNA Phe T=44 °
GAIN IN I N T E N S I T Y W I T H Mg (e) LOSS (')
yeast tRNA Phe T=44 °
,]"/ ~,
3 Spermine/tRNA/
I
]
]
]6
15
14
1___i 15
12
L
I[
_ I I0
15
I
14
J
15
L _ _ ~ _ ppm
12
It
Fig. 5. E f f e c t o f m a g n e s i u m o n t h e 3 0 0 M H z N M R s p e c t r u m o f y e a s t t R N A Phe at 4 4 ° C in t h e p r e s e n c e o f 3 s p e r m i n e / t R N A . See Fig. 1 f o r a d d i t i o n a l details.
17 TABLE I EFFECT OF POLYVALENT
CATIONS ON tRNA STRUCTURE
Polyvalent cation
Temperature (o C)
Nos. of cations w h i c h stabilize additional hydrogen bonds
Number of additional base p a i r s stabilized
Type of stabilization
Mg 2+ * Mg 2+ * Mg 2+ *
22 37 44
2 2 4
Spermine * Mg 2+ **
44 44 (3 s p e r m i n c / tRNA)
3 2
1 2 4 2 2
semi-sequential sequential sequential sequential sequential
-+ 1 -+ 1 -+ 1 + 1 -+ 1
* t R N A c o n c e n t r a t i o n ~ 1 m M , t o t a l s o d i u m c o n c e n t r a t i o n ~ 0 . 1 7 M. ** S a m e as in *, w i t h 3 s p e r m i n e / t R N A , see t e x t for details.
The polyamine spermine stabilizes t R N A structure in a manner somewhat different from the stabilization by magnesium. As the results in Table I show, spermine is less effective than magnesium in stabilizing t R N A structure and comparison of the spectra in Figs. 3 and 4 show that spermine and magnesium effect the spectrum of yeast t R N A Phe in different ways. For example, the addition of just one magnesium induces the gain of intensity near 14.4 ppm, whereas spermine does not. Some of the other changes induced by spermine are similar, however, to those induced by magnesium. Thus, spermine appears to stabilize some but not all of the structural features stabilized by magnesium. However, it was found that the combination of spermine and magnesium, at low levels, is more effective than either cation alone in stabilizing t R N A structure. In addition the combination of 3 spermine/tRNA and 2 Mg2+/tRNA completely stabilizes t R N A structure at 44 ° C. Thus it appears that spermine alone cannot stabilize the native t R N A structure at 44°C but in combination with magnesium spermine is effective in stabilizing t R N A structure. Comments The results in Table I show that the number of magnesium required to stabilize the native t R N A structure is dependent on the exact experimental conditions t. This may accound for the apparent discrepancy between the results of Stein and Crothers [2,3] and those of RSmer and Hach [1]. R5mer and Hach [1] examined the binding of magnesium to t R N A in the presence of 0.032 M Na ÷ from 10 to 45°C and observed five strong binding sites with K ~ 10 s M -1. Stein and Crothers [2,3] examined t R N A in 0.17 M Na ÷ at 4°C and found a
T T h e s p e c t r a s h o w n h e r e for y e a s t t R N A Phe in the a b s e n c e o f m a g n e s i u m are nearly i d e n t i c a l t o t h o s e o b t a i n e d p r e v i o u s l y b y Hflbers et al. [ 2 3 ] b u t d i f f e r s i g n i f i c a n t l y f r o m t h o s e o f RobUlard e t al. [ 2 0 - - 2 2 ] w h o f o u n d n o e f f e c t o f m a g n e s i u m o n the s p e c t r u m o f y e a s t t R N A P h e in the t e m p e r ature range e x a m i n e d here. T h e fact t h a t Robillard et al. [ 2 0 - - 2 2 ] did n o t o b s e r v e a m a g n e s i u m e f f e c t i n d i c a t e s that their t R N A s a m p l e s c o n t a i n e d m a g n e s i u m , a l t h o u g h t h e y w e r e p r e s u m e d t o be free o f m a g n e s i u m . T h e i r p r o c e d u r e for r e m o v a l o f m a g n e s i u m ( h e a t i n g o f t R N A , ~ 1 r a M , in the p r e s e n c e o f E D T A , 10 r a M ) will n o t r e m o v e m a g n e s i u m f r o m t R N A s i n c e the binding c o n s t a n t o f m a g n e s i u m to E D T A is q u i t e s m a l l , K ~ 105 [ 1 ] .
18 Single strong binding site with K ~ 104 M -'. The difference in binding constants and number of sites can be attributed to competition between sodium and magnesium for the strong binding sites as well as stabilization of t R N A structure by sodium. As the t R N A structure is stabilized the number and strength of binding sites are reduced as a result of a reduction in the free energy gained by the binding of magnesium. The NMR results presented here have allowed us to determine the number of magnesium which stabilize tRNA structure in different experimental conditions, the number of base pairs stabilized by magnesium, determination of some of the structural features effected by magnesium, and the relation between the stabilization of t R N A by magnesium and by polyamines. The results are in general agreement with those presented elsewhere, but in several instances, for example, the effect of magnesium on the structure of tRNA at low temperature in the presence of high levels of sodium offer information unobtainable by other techniques. Additional information can be obtained through an examination of the effect of magnesium on the spectra of other tRNA, as well as other spectral regions, and such studies are underway. Acknowledgements
The support of the U.S. Public Health Service (Grant 22969) is most gratefully acknowledged. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
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