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Helix-coil dynamics in RNA: The amino acid acceptor helix of Escherichia coli phenylalanine transfer RNA
(1980) 142, 181-193
J. Mol. Biol.
Helix-Coil Dynamics in RNA : The Amino Acid Acceptor Helix of Escherichiu coli Phenylalanine Transfer RNA RALPH E. HURD AND BRIAN R. REID Department of Biochemistry University of California Riverside, Calij’. 92521, U .S.A . (Received 15 November 1979, and in revised form 2 May 1980) The RNA helix is a fluctuating dynamic structure in solution. In this paper we report a kinetic description of the individual base-pairs in an RNA helix (the acceptor stem of Escherichia coli tRNAPhB) obtained by proton nuclear magnetic resonance spectroscopy using the saturation recovery techniques described by Johnston & Redfield (1977). We have determined the helix opening rates of each of the six Watson-Crick G*C base-pairs in the acceptor stem of partially unin the presence of buffers, under folded E. coli tRNAPhe at elevated temperature which conditions saturation recovery is exchange-dominated and the exchange process is rate-limited by helix-coil opening.
1. Introduction (CW) nuclear magnetic resonance methods, Kearns et al. (1971a,b) first observed the exchangeable ring NH protons of complementary base-pairs (guanosine NlH and uridine N3H) using approximately 1 mM solutions of transfer RNA in water. These resonances are found between - 11 p.p.m.t and -15 p.p.m. from 2,2 dimethylsilapentane-5-sulfonate and are only observed for base-pairs with helix lifetimes of 5 ms and longer. More recently, Johnston & Redfield (1977) reported the use of a modified long observation pulse (the Redfield 21412 pulse) to selectively excite the low field region of the tRNA spectrum. The frequency transform of a long pulse falls to zero close to the reciprocal of the pulse length, and this null is set at the water resonance frequency to avoid, or at least greatly reduce, the dynamic range problems caused by the enormous solvent resonance. The major advantage of this pulse Fourier transform method is the ability to carry out timeresolved nuclear magnetic resonance spectroscopy. Using the 21412 observation pulse following selective saturation of the various spectral peaks, Johnston and Redfield studied the saturation recovery rates of the 27% 1 base-pair ring NH protons in yeast tRNAPhe at temperatures between 10°C and 45°C. A problem encountered in these 270 MHz studies was the inability to determine individual proton rates from the saturation recovery of complex unresolved peaks; even at 360 MHz, we find a peak of seven unresolved resonances in the low field spectrum of yeast tRNAPhe.
Using
conventional
t Abbreviation
frequency-sweep
used: p.p.m., parts per million. 181
0022-2836/80/260181-13
$02.00/O
0 1980 Academic
Press Inc. (London)
Ltd.
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This lack of resolution makes it difficult. to carry out a complete dynamic analysis of any single helix in the tRNA structure. There is not only the uncertainty in extracting individual rates from a multiphasic recovery, but there is also an uncertainty in assigning a specific base-pair to a particular rate once it has been determined. A further complication is that the dynamics of helix-coil transitions are relatively slow around physiological temperature, especially in the presence of stabilizing magnesium ions. Under such conditions the mechanism of recovery from saturation is no longer predominantly governed by exchange and becomes dominated by purely magnetic spin-lattice relaxation processes. This makes the determination of the helix opening and chemical exchange rates much more difficult. In our previous studies on Escherichia coli tRNA Phe we noted that 21 of the 27 basepair resonances could be eliminated from the spectrum by partially unfolding the molecule at 68°C in the presence of magnesium, leaving six low field resonances from the consecutive G.C pairs in the acceptor stem (Reid & Hurd, 1977). We have repeated this experiment in the absence of magnesium; under these conditions the intensity of the spectrum at 35°C is 27*1 protons which simplifies to the same six sharp G*C resonances at temperatures in the range from 52°C to 65”C, i.e. the molecule is less stable but the acceptor helix remains the most stable part of the molecule. A broad resonance assigned to AU7, the terminal base-pair of the acceptor stem, is present at 52°C but disappears completely by 62°C. In this paper we take advantage of the differential thermal stability of the acceptor stem of E. coli tRNAPhe to determine the exchange-dominated saturation recovery behavior of each peak in the simplified six-resonance spectrum at elevated temperature, from which we present a dynamic picture of this RNA helix.
2. Materials and Methods E.
purified to homogeneity (1850 pmol phenylalanine/./I,,, unit) by chromatography of crude E. coli B tRNA on BD-cellulose (Gillam et al., 1967) followed by DEAE-Sephadex (Nishimura, 1971) and Sepharose 4B chromatography (Holmes et al., 1975). Reagent grade chemicals were used in all stages. A total of 7 mg (140 Azeo units) of pure water-dialyzed, lyophilized E. coli rRNAPhe was dissolved in 0.2 ml of appropriate buffer and transferred to a Wilmad 508CP nuclear magnetic resonance microtube. Spectra were obtained at 360 MHz, in the pulse-FT mode, at the Stanford Magnetic Resonance Laboratory. Pre-irradiation of individual resonances was carried out for 100 ms or 200 ms using enough radio frequency power to just saturate the peak in question during the pre-irradiation pulse. The spectra were collected at various time delays (1 ms to 1 s) after switching off the pre-irradiation using a Redfield 21412 observation pulse of 370 ps which was centered at 2628 Hz downfield of the water resonance frequency. The pre-irradiation-delay-observation pulse sequenoe w&s repeated for 2 or 3 blocks of 512 transients which were Fourier transformed and block-averaged to improve signal-to-noise ratios. COG
tRNAPhe
W&S
3. Results the low field spectrum of E. c&i temperatures and that upon partially unfolding the molecule at 68”C, only the six consecutive G-C pairs in the acceptor stem remain detectable in the spectrum (Reid & Hurd, 1977). We have shown that in the presence of magnesium
tRNAPhe
contains
resonances
from
27 base-pairs
at physiological
HELIX-COIL
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DYNAMICS
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RNA
L---xlljd
J’LA
‘LA-“----/ I1
IN
I
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p.p.m. FIG. 1. The low field
proton nuclear magnetic resonance spectra of E. coli tRNAPhB in 5 musodium phosphate (pH 7.0), 100 mu-N&l at 51”C, 53”C, 55”C, 57”C, 61°C and 65°C. Assignments of GCl, CG2, CG3, CG4, GC5, GC6 and AU7 are discussed in the text and indicated by number under the 51°C spectrum. The resonance at -9 p.p.m. belongs to the C8H of m’G46. Intensity is lost,, especially for GCl and GC6, by 65°C.
This same unfolding transition can be brought about at a considerably lower temperature in the absence of magnesium. The spectra of E. coli tRNAPhe dissolved in 5 mM-sodium phosphate buffer (pH 7.0) containing 100 mM-sodium chloride are shown in Figure 1 at temperat,ures from 51°C to 65°C. Based on linewidth, chemical modification studies and its non-exchangeability (on a time-scale of several minutes) we have shown that the resonance located at about -9 p.p.m. belongs to the C8H of m’G46 (Hurd & Reid, 1979). In the extreme low field region the observed resonance positions are in excellent agreement with the predicted chemical shifts of the acceptor helix based on the nearest-neighbor and next to nearest-neighbor ring current shifts calculated by Arter & Schmidt (1976) for an 11-fold helix combined with a value of - 13.45 p.p,m. for the unshifted starting position of a G-C base-pair (Reid et al., 1979). The observed/predicted positions are as follows : GCl (1255/12.52); CG2 (13.17/13.10); CG3 (12.70/12.79); CG4 (12.55/ 12.50); GC5 (12.38/12.46); GC6 (12*25/12*31). The resonance at about -13.7 p.p.m. is derived from the terminal base-pair AU7 at the bottom of the acceptor helix; it is severely broadened at 51 “C and broadens beyond detection above 55°C due to solvent exchange at rates above 200 s-l. Transfer of saturation from water to the six G.C base-pair resonances during 200 ms of water pre-irradiation (1 ms delay before the observation pulse) was carried out in a preliminary experiment (data not shown). As expected, the very slowly exchangeable aromatic C8H proton of m”G46 is relatively unaffected, whereas more than 90% of every base-pair resonance disappears when water is saturated due to base-pair exchange with the saturated water protons. This indicates that the proton exchange rate is at least 5 s-l.
184
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AND
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p.p.m. FIG. 2. (pH 7.0), times of resonance
Saturation recovery of the CG2 base-pair resonance at 58°C in 5 mna-sodium phosphate 100 mix-NaCl. The spectra were obtained with a 380 ps Redfield 21412 pulse after delay 1 ms, 10 ms, 20 ms, 50 ms and 100 ms following a 200 ms saturation pulse at the CG2 frequency.
Figure 2 shows the recovery from saturation of base-pair CG2 at 58°C in 5 mMsodium phosphate (pH 7-O), 100 m&r-sodium chloride. The spectra were obtained using a 200 ms pre-irradiation pulse centered at -13.17 p.p.m. to saturate the CG2 resonance, then a variable delay (1 ms to 500 ms) followed by a 0.38 ms Redfield 21612 observation pulse. Individual spectra required between five and ten minutes of signal averaging which was obtained as three separate blocks of averaged time domain data; these were Fourier transformed, phased, added, then plotted after baseline correction. Saturation recovery was also studied as a function of temperature. Figure 3 shows the data for CG3 at 52°C and 62°C. The saturation recovery rates for single resonances, such as CG3, were determined from the slope of the logarithm of fractional saturation
HELIX-COIL
DYNAMICS
IN
RNA
150 ms
I
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I1
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I1
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11
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p.p.m. FIG. 3. Saturation acquired as described
recovery of the CG3 base-pair resonance at 52°C in the legend to Fig. 2 using the same sample.
and
62’C.
Spectra
were
remaining versus delay time. Semi-logarithmic plots of the saturation recovery of CG3 (-12.7 p.p.m.) at 52”C, 58°C and 62°C in 5 mM-sodium phosphate (pH 7-O), 100 mwsodium chloride are given in Figure 4. The y-intercepts of the 58°C and 62°C link are near 1.0 indicating nearly complete saturation at time zero. The approximately 0.81 y-intercept of the 52°C line does not necessarily indicate only 81 y0 saturation of
P.P.m
0.0, L
50
100 150 200 Delay time (msl
Fm. 4. The saturation recovery rate plots for CG3 at 52”C, 58°C and 62’C presented aa fractional saturation ver~u8 delay time. The slope of the natural logarithm of fraction recovered 2rewus delay time gives the following rates: 52’C (9 s-l), 58°C (18 8-l) and 62°C (35 s-l).
186
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B.
k open
R.
REID
ki [cat.]
rclose FIG. 5. A schematic diagram coil and water, axe shown.
of proton
exchange
from
the helix.
The
3 states
involved,
helix,
the CG3 resonance, however, since a small amount of a broadened base-pair from the anticodon helix is still present (albeit exchanging very rapidly) at 52°C under these solvent conditions (note the relative intensities at -12.7 p.p.m. and -12.4 p.p.m. between 51°C and 55°C compared to their relative intensities between 55°C and 61°C in Fig. 1). Between 52°C and 62°C the saturation recovery rate of the CG3 base-pair resonance increases about fourfold (from 9 s-l to 35 s - l). The relationship between logarithm rate and reciprocal temperature yields a non-linear Arrhenius plot for the three temperature points studied. The reasons for this probably lie in the dual mechanism of recovery from saturation. The two mechanisms are (1) exchange with an unsaturated water proton, and (2) the purely magnetic spin-lattice relaxation; the experimentally observed saturation recovery rate is the arithmetic sum of these two processes. The exchange process, whatever its mechanism, presumably has a specific activation energy and therefore a linear Arrhenius plot and hence we empirically sought a constant which, when subtracted from each data point, produced a linear plot. A value of about 6 s- 1 satisfied this criterion and we tentatively equate this rate as the approximate spin-lattice relaxation rate. Slthough the spin-lattice rate is not expected to be temperature-independent, it is expected to be relatively small and its temperature variation across the 10 deg. range we have studied is probably less than the combined errors in spectral peak integration and in the Arrhenius plot. Using this value together with the saturation recovery rate leads to proton exchange rates of 3 s-l at 52°C and 30 s-l at 62”C, i.e. a surprising tenfold increase in exchange rate over a 10 deg. range. The ring NH protons studied here are located in the core of the RNA helix and are inaccessible to direct exchange with water. Hence proton exchange must involve two separate processes, namely (1) a local structural opening or “breathing” followed by (2) chemical exchange from the exposed coil state. These processes are shown schematically in Figure 5. An important and interesting property to know would be the breathing or opening rate, k,,,,, for a single base-pair in a given helix. Such information for each individual base-pair would then permit a detailed analysis of the extent to which the helix-coil transition was fully co-operative throughout that helix. However, the observed proton exchange rate is not’ necessarily the helix opening rate, especially when the coil-water rate, k.[cat.], is the same or less than k open;only when k.[cat.] is very fast will the observed exchange rate be equal to, and rate-limited by, the rate of helix opening. The purely chemical coil-water rate, k.[cat.], is buffer-catalyzed and dependent upon pH, type of buffer and concentration
HELIX-COIL
DYNAMICS
IN
GCI CG4 1
RNA
187
GCI 7”
130 ms
I,
-15
I
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,
I
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I,
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I I I I I1
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and
CG4
I I I -10
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p.p.m. FIG. 6. Saturation sodium
phosphate
(pH
recovery of the 2.proton 7.0), 100 mm-NaCl.
peak
containing
GCl
at 62°C
in 5
IKIM-
of buffer (Kallenbach et al., 1976; Eigen, 1964); hence k.[cat.] can be experimentally manipulated via pH and buffer concentration and, if this step is limiting in the exchange process, such changes should dramatically affect both the exchange rate and the observed saturation recovery rate. We therefore repeated the saturation recovery experiments at various pH values, various buffer concentrations, and with additional buffers ; the results of buffer addition and pH changes under isoionic conditions are summarized in Figure 11. At 63°C and pH 5.8 the saturation recovery rate of CG3 is 13 s-l in 5 mw-sodium phosphate buffer and increases only slightly to 15 s-l in 50 mlcl-sodium phosphate buffer (total Na+ was adjusted to 408 mM in both cases by addition of N&l). The rate st pH 7.2 in 5 mM-phosphate is unaffected by the addition of 10 mnn-Trisa HCl. Lastly, instead of decreasing upon reduction of [OH-], the saturation recovery rate increases slightly at pH values below neutrality. These results conclusively demonstrate that proton exchange is not limited by the coil-water exchange rate (expect,ed to be ab least lo5 s-l in 10 rnM-Tris; see Kallenbach et al., 1976) ; hence the helix opening rate, k,,,,, must be rate-limiting in the exchangedominated saturation recovery. The small increase in saturation recovery rate as the pH is lowered must therefore reflect a slight destabilization (faster k,,,,) under acidic conditions. A similar result was observed for the adjacent base-pair CG2. Using tritium isotope methods, Teitelbaum & Englander (1975) showed that proton exchange into the ring NH of G * C helices is open-limited at low temperature ; the same appears to be true at 63°C if adequate buffer catalysis is provided to maintain rapid coil-water
188
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300 ms -
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---
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(a)
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(‘b)
FIG. 7. Saturation recovery of CG4 aa a single resonance at 52°C. (a) Effect of increasing Coa+ on the spectrum of E. coli tRNAPhe m 5 mmsodium phosphate (pH 7.0), 100 mu-NaCl at 62% Titration with Coa+ to 260 ELM shifts GC6 (0.26 p,p.m.), CC1 (0.17p.p.m.) and CC2 (@07p.p.m.) upfield. CC1 is shifted away from CG4 onto CC%. (b) The saturation recovery of the single CG4 resonance exposed by Co2 + .
exchange. After spin-lattice correction the saturation recovery rate can now be directly equated with k,,,,, and hence the activation energy for opening the helix at that particular base-pair can now be calculated from the temperature dependence; the activation energy for opening CG3 appears to be approximately 49 kcal/mol.
0.1 1
FICZ. 8. The semi-logarithmic data were obtained at 62°C N&l and 0.26 mM-CoCl,.
I 50
I 100
I 150
plot of the saturation on a sample containing
I 200
I 250
1 300
recovery rate of the CG4 resonance. These 6 mM-sodium phosphate (pH 7.0), 100 mM-
HELIX-COIL
DYNAMICS
IN
RNA
189
62’ C1
58O (
h
5
6
12 s-
73 s-1
I 100
1 50
I 150
I 200 T hs)
FIG. 9. Semi-logarithmic Samples
were dissolved
plots of GC5 (0) and GC6 (0) saturation recovery in 5 mm-sodium phosphate (pH 7,0), 100 nm-NaCl.
at 58°C and
62°C.
Recovery of the two unresolved resonances, GCl and CG4, at 62°C is shown in Figure 6. These data were obtained in 5 mM-sodium phosphate (pH 7.0), 100 mMsodium chloride. From simple inspection it is obvious that an intensity equivalent to one proton is rapidly recovered in the first 20 ms whereas the second proton requires about 100 ms for full recovery. The semi-logarithmic plots for this two-proton peak (not shown) revealed distinctly biphasic recovery from which the slower rate was found to be 21 s-l. Correcting for the contribution of the slower rate we determined we deduce that the slower the rate of the faster proton to be about 90 s- I. Intuitively rate is the recovery rate of the interior base-pair CG4, and that the faster rate is associated with the terminal base-pair GCl.
-15
-14
-13
-12
-II
-10
-9
-15
-14
-13
-12
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-10
-9
P4.m FIG. 10. Saturation phosphate
(pH
7.0),
recovery of the m’G46 100 miwNaC1.
C8H
resonance
at 58°C
and
62°C
in 5 mm-sodium
190
R.
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HURD
AND
Saturation
B.
R.
REID
recovery rates (s-l)
&OH
FIG. 11. Summary G *C base-pairs under
of saturation recovery a variety of conditions.
rates
determined
for
the ring
NH
protons
of the
6
Addition of Co2+ to the sample (80 PM to 260 PM) in 5 miw-sodium phosphate (pH 7-O), 100 mM-sodium chloride at 52°C shifts the terminal and penultimate basepair resonances upfield (GCl, GC6 > CG2) ; during this titration GCl is upfield shifted onto the GC5 resonance thus exposing CG4 as a single resonance. The saturation recovery of CG4 at 52°C under these conditions is shown (along with the Co2+ titration) in Figure 7. A semi-logarithmic plot of the data is shown in Figure 8 ; it is a single exponential. The rate, 6 s-l, is consistent with the other rates determined at 52°C. The exchange rate of neighboring base-pairs in a helix can be quite different as shown in Figure 9 in which the saturation recovery rates of GC5 and GC6 are plotted together at two temperatures. At both 58°C and 62”C, there is a more than fourfold difference in the recovery rates for these two adjacent base-pairs. The helix opening rate for AU7 is so rapid (>200 s- ‘) that its resonance is not even observed at 58°C (see Fig. 1). The saturation recovery rate was also determined for the non-exchangeable (at least on this time-scale) aromatic C8H of mlG46 at 58°C and 62°C and the data are shown in Figure 10. These rates, 1 s- l at 58°C and 1.4 s - l at 62”C, are presumably dominated by magnetic spin-lattice relaxation processes with very little, if any, exchange component. The increase in the spin-lattice relaxation rate from about 1 to 1.5 s-l for the largely random coil m7G46 C8H to about 6 s-l for the structured, hydrogen-bonded proton of CG3 is not unreasonable considering the increased relaxation contributed by its association with two nitrogen atoms.
HELIX-COIL
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191
4. Discussion The saturation recovery rates obtained for the six G. C base-pair resonances from the E. wli tRNAPhe acceptor stem are summarized in Figure 11. Exchange-in of saturated water protons was shown to be greater than 90% within 200 ms at 58°C. This, combined with the observation that the saturation recovery rates are highly t’emperature-dependent, indicated that the recovery process for all resonances is dominated by exchange with water protons. The buffer and pH studies clearly indicate that under these conditions the saturation recovery rates are independent of the coil-water exchange rate and reflect the different helix opening rates for individual base-pairs (combined with a much slower magnetic recovery rate). An interesting observation is that adjacent base-pairs can have quite large differences in their helix opening rates. For instance at 58°C the recovery rate of GC5 is 18 s - l (helix opening approx. 6 s- ‘) whereas GC6 recovers at 73 s - l (helix opening approx. 67 s-l) i.e. differences of approximately tenfold for nearest-neighbors at the distal end of the helix. At the open end of the helix the breathing rates (after subtracting the estimated non-exchange relaxation) are about 35 s-l for GCl and about 16 se1 for CG2 at 58°C. The small uncertainty in the precise value of the breathing rate derives from our lack of knowledge concerning the mechanism, and therefore the temperature dependence. of the non-exchange component of the relaxation. We estimate the nonexchange relaxation to have an average value of about 6 s-l across the 52°C to 62°C t,emperabure range. The classical dipolar relaxation rate for the helix will be somewhat IPSS than that of the more flexible m7G C8H (1 s-l at 58°C) and, at 360 MHz, will show a mild correlation time-dependent increase at 62’C, since the correlation time of the partially unfolded tRNA must still be well above 3 x lo-lo s. Assuming no gross structural unfolding across this temperature range, the increase in the rate of normal dipolar relaxation will be less than 20%, i.e. less than 1.2 s-l. The remaining 4 to 5 s-l of non-exchange relaxation presumably occurs via a combination of spindiffusion and quadrupolar relaxation of the ring NH protons. Relaxation via spindiffusion (Stoesz et al., 1978) is expected to decrease as the rotational correlation time is reduced by raising the temperature, and is not expected to be significant for partially unfolded molecules around 58°C (Stoesz et al. (1978) estimate this crossrelaxation rate to be about 4 s-l at 20°C for histidine NH protons in a polymer of similar size to fully structured tRNA). While spin-diffusion would complicate exchange analysis studied by transfer of saturation from pre-irradiated water to ring NH protons, it is less of a problem when the inherently rapidly relaxing ring NH protons are selectively irradiated as we have done. Hence probably the major non-exchange relaxation is via the rapid quadrupolar relaxation of the two nitrogen atoms between w,hich the ring NH proton is shared in the hydrogen-bonded state. This process is expectted to have a weak temperature dependence similar to that of dipolar relaxation. in the non-exchange relaxation These unknown effects introduce a level of uncertainty rate H hich must be subtracted from the saturation recovery rates to obtain the true breathing rates. However, a conservative estimat,e of these errors still leads to nonexchange recovery rates of 652 s- l. In the case of observed saturation recovery rates of 25 s- l or higher the error in the estimated breathing rates is less than * lo:/, which is approaching the precision of measuring peak heights at the S/N ratios in
192
R.
E. HURD
AND
B. R.
REID
our spectra. However, this error becomes more serious at slow saturation recovery rates, e.g. the rate of 9 s-l for CG3 at 52°C could correspond to breathing rates anywhere from 1 s-l to 5 s-l. For these reasons we have refrained from attempting to extract accurate activation energies for breaking each individual base-pair. There is a qualitative sequential trend to higher activation energies towards the interior of the helix (with the exception of GCl which appears to remain stacked with A73), which is consistent with obligatory dissociation of exterior base-pairs before internal ones can breathe; however, rigorous corroboration of this idea requires much more extensive data than that presented here. An obvious application of this technique is the analysis of the dynamics of each secondary and tertiary base-pair in a native tRNA molecule but there are several problems which complicate such an analysis, not the least of which is the increased ambiguity in precise assignment of overlapping resonances in crowded, complex 27-line spectra. However, the approach can be turned around and used to assist in the determination of which resonances are derived from tertiary interactions based on their kinetic behavior, as has been elegantly demonstrated by Johnston & Redfield (1977). A further complication is that processes other than exchange dominate the recovery process at lower temperatures. Johnston & Redfield (1978) have pointed out that only when saturation recovery rates become st,rongly temperature-dependent at rates above about 5 s - l can useful exchange dynamics be extracted from the data ; however, they also showed that such conditions could be met at close to physiological temperatures by appropriate manipulation of the solvent. A more conservative aim towards which we are working is the effect of permuted base sequences, including G.U pairs, on local dynamics and stability in short RNA helices such as the one described here. Such investigations are carried out at relatively high temperatures where the recovery rates are heavily dominated by exchange and under buffer conditions where exchange is open-limited. Changing the sequence of the six G * C and C. G pairs, as well as the effects of G-U or A. U replacements, will hopefully shed some light on the fundamental principles underlying the sequence dependence of the stability of RNA helices. The authors gratefully acknowledge the use of the Stanford Magnetic Resonance Laboratory (supported by National Science Foundation grant GR23633 and by National and Lillian Institutes of Health grant RR0071 1). 0 ur sincere thanks go to Susan Ribeiro McCollum for their expertise in purifying the E. coli phenylalanine tRNA used in this study. We are especially indebted to Edward Azhderian for his help in obtaining some of the spectra which were presented. We also thank Edward Azhderian and Dr Do-Lan HOO for stimulating discussions on theoretical aspects of this study. This research was SUPported by grants (to B.R.R.) from the National Science Foundation (PCM-7902904), the National Institutes of Health (GM/CA 27005) and the American Cancer Society (NP-191).
REFERENCES Arter, D. B. & Schmidt, P. G. (1976). Nucl. Acids Res. 3, 1437-1447. Eigen, M. (1964). Angew Chem. Int. Ed. England, 13, 1-19. Gillam, I., Millward, S., Blew, D., Van Tigerstrom, M., Wimmer, E. & Tener, G. M. (1967). Biochemistry, 6, 3043-3056. Holmes, W. M., Hurd, R. E., Reid, B. R., Rimerman, R. A. & Hatfield, G. W. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 1068-1071.
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Hurd, R. E. & Reid, B. R. (1979). Biochemistry, 18, 4017-4024. .Johnston, P. D. & Redfield, A. G. (1977). Nucl. Acids Res. 4, 3599-3615. Johnston, P. D. & Redfield, A. G. (1978). Nucl. Acids Re.s. 5, 3913-3927. Kallenbach, N. R., Daniel, W. E. & Kaminker, M. A. (1976). Biochemistry, 15, 121%1228. Kearns, D. R., Patel, D. & Shulman, R. G. (1971a). Nature (London), 229, 338-340. Kearns, D. R., Patel, D., Shulman, R. G. & Yamane, T. (19715). J. MoZ. Biol. 61, 265270. Nishimura, S. (1971). Procedures in Nucleic Acid Research (Cantoni, G. L. & Davies, D. R., eds), vol. 2, pp. 5422564, Harper and Row, New York. Reid, B. R. & Hurd, R. E. (1977). Act. Chem. Res. 10, 396-402. Reid, B. R., McCollum, L., Ribeiro, N. S., Abbate, J. & Hurd, R. E. (1979). Biochemistry, 18, 39964004. Stoesz, J. D.. Redfield, A. G. & Malinowski, D. (1978). FEBS Letters, 91, 320-325. Teitelbaum. H. & Enplander, S. W. (1975). J. Mol. BioZ. 92, 79-92.
J. Mol. Biol.
Helix-Coil Dynamics in RNA : The Amino Acid Acceptor Helix of Escherichiu coli Phenylalanine Transfer RNA RALPH E. HURD AND BRIAN R. REID Department of Biochemistry University of California Riverside, Calij’. 92521, U .S.A . (Received 15 November 1979, and in revised form 2 May 1980) The RNA helix is a fluctuating dynamic structure in solution. In this paper we report a kinetic description of the individual base-pairs in an RNA helix (the acceptor stem of Escherichia coli tRNAPhB) obtained by proton nuclear magnetic resonance spectroscopy using the saturation recovery techniques described by Johnston & Redfield (1977). We have determined the helix opening rates of each of the six Watson-Crick G*C base-pairs in the acceptor stem of partially unin the presence of buffers, under folded E. coli tRNAPhe at elevated temperature which conditions saturation recovery is exchange-dominated and the exchange process is rate-limited by helix-coil opening.
1. Introduction (CW) nuclear magnetic resonance methods, Kearns et al. (1971a,b) first observed the exchangeable ring NH protons of complementary base-pairs (guanosine NlH and uridine N3H) using approximately 1 mM solutions of transfer RNA in water. These resonances are found between - 11 p.p.m.t and -15 p.p.m. from 2,2 dimethylsilapentane-5-sulfonate and are only observed for base-pairs with helix lifetimes of 5 ms and longer. More recently, Johnston & Redfield (1977) reported the use of a modified long observation pulse (the Redfield 21412 pulse) to selectively excite the low field region of the tRNA spectrum. The frequency transform of a long pulse falls to zero close to the reciprocal of the pulse length, and this null is set at the water resonance frequency to avoid, or at least greatly reduce, the dynamic range problems caused by the enormous solvent resonance. The major advantage of this pulse Fourier transform method is the ability to carry out timeresolved nuclear magnetic resonance spectroscopy. Using the 21412 observation pulse following selective saturation of the various spectral peaks, Johnston and Redfield studied the saturation recovery rates of the 27% 1 base-pair ring NH protons in yeast tRNAPhe at temperatures between 10°C and 45°C. A problem encountered in these 270 MHz studies was the inability to determine individual proton rates from the saturation recovery of complex unresolved peaks; even at 360 MHz, we find a peak of seven unresolved resonances in the low field spectrum of yeast tRNAPhe.
Using
conventional
t Abbreviation
frequency-sweep
used: p.p.m., parts per million. 181
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Press Inc. (London)
Ltd.
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This lack of resolution makes it difficult. to carry out a complete dynamic analysis of any single helix in the tRNA structure. There is not only the uncertainty in extracting individual rates from a multiphasic recovery, but there is also an uncertainty in assigning a specific base-pair to a particular rate once it has been determined. A further complication is that the dynamics of helix-coil transitions are relatively slow around physiological temperature, especially in the presence of stabilizing magnesium ions. Under such conditions the mechanism of recovery from saturation is no longer predominantly governed by exchange and becomes dominated by purely magnetic spin-lattice relaxation processes. This makes the determination of the helix opening and chemical exchange rates much more difficult. In our previous studies on Escherichia coli tRNA Phe we noted that 21 of the 27 basepair resonances could be eliminated from the spectrum by partially unfolding the molecule at 68°C in the presence of magnesium, leaving six low field resonances from the consecutive G.C pairs in the acceptor stem (Reid & Hurd, 1977). We have repeated this experiment in the absence of magnesium; under these conditions the intensity of the spectrum at 35°C is 27*1 protons which simplifies to the same six sharp G*C resonances at temperatures in the range from 52°C to 65”C, i.e. the molecule is less stable but the acceptor helix remains the most stable part of the molecule. A broad resonance assigned to AU7, the terminal base-pair of the acceptor stem, is present at 52°C but disappears completely by 62°C. In this paper we take advantage of the differential thermal stability of the acceptor stem of E. coli tRNAPhe to determine the exchange-dominated saturation recovery behavior of each peak in the simplified six-resonance spectrum at elevated temperature, from which we present a dynamic picture of this RNA helix.
2. Materials and Methods E.
purified to homogeneity (1850 pmol phenylalanine/./I,,, unit) by chromatography of crude E. coli B tRNA on BD-cellulose (Gillam et al., 1967) followed by DEAE-Sephadex (Nishimura, 1971) and Sepharose 4B chromatography (Holmes et al., 1975). Reagent grade chemicals were used in all stages. A total of 7 mg (140 Azeo units) of pure water-dialyzed, lyophilized E. coli rRNAPhe was dissolved in 0.2 ml of appropriate buffer and transferred to a Wilmad 508CP nuclear magnetic resonance microtube. Spectra were obtained at 360 MHz, in the pulse-FT mode, at the Stanford Magnetic Resonance Laboratory. Pre-irradiation of individual resonances was carried out for 100 ms or 200 ms using enough radio frequency power to just saturate the peak in question during the pre-irradiation pulse. The spectra were collected at various time delays (1 ms to 1 s) after switching off the pre-irradiation using a Redfield 21412 observation pulse of 370 ps which was centered at 2628 Hz downfield of the water resonance frequency. The pre-irradiation-delay-observation pulse sequenoe w&s repeated for 2 or 3 blocks of 512 transients which were Fourier transformed and block-averaged to improve signal-to-noise ratios. COG
tRNAPhe
W&S
3. Results the low field spectrum of E. c&i temperatures and that upon partially unfolding the molecule at 68”C, only the six consecutive G-C pairs in the acceptor stem remain detectable in the spectrum (Reid & Hurd, 1977). We have shown that in the presence of magnesium
tRNAPhe
contains
resonances
from
27 base-pairs
at physiological
HELIX-COIL
__-. _ ^ _.ril, I,
I
-15
-14
‘1 I
DYNAMICS
-13
I
-12
I
183
RNA
L---xlljd
J’LA
‘LA-“----/ I1
IN
I
-II
I
I
-10
L
I
I
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I
I1
I
-14
-13
I
I1
I
-12
-II
I
I
-10
I
I
-9
p.p.m. FIG. 1. The low field
proton nuclear magnetic resonance spectra of E. coli tRNAPhB in 5 musodium phosphate (pH 7.0), 100 mu-N&l at 51”C, 53”C, 55”C, 57”C, 61°C and 65°C. Assignments of GCl, CG2, CG3, CG4, GC5, GC6 and AU7 are discussed in the text and indicated by number under the 51°C spectrum. The resonance at -9 p.p.m. belongs to the C8H of m’G46. Intensity is lost,, especially for GCl and GC6, by 65°C.
This same unfolding transition can be brought about at a considerably lower temperature in the absence of magnesium. The spectra of E. coli tRNAPhe dissolved in 5 mM-sodium phosphate buffer (pH 7.0) containing 100 mM-sodium chloride are shown in Figure 1 at temperat,ures from 51°C to 65°C. Based on linewidth, chemical modification studies and its non-exchangeability (on a time-scale of several minutes) we have shown that the resonance located at about -9 p.p.m. belongs to the C8H of m’G46 (Hurd & Reid, 1979). In the extreme low field region the observed resonance positions are in excellent agreement with the predicted chemical shifts of the acceptor helix based on the nearest-neighbor and next to nearest-neighbor ring current shifts calculated by Arter & Schmidt (1976) for an 11-fold helix combined with a value of - 13.45 p.p,m. for the unshifted starting position of a G-C base-pair (Reid et al., 1979). The observed/predicted positions are as follows : GCl (1255/12.52); CG2 (13.17/13.10); CG3 (12.70/12.79); CG4 (12.55/ 12.50); GC5 (12.38/12.46); GC6 (12*25/12*31). The resonance at about -13.7 p.p.m. is derived from the terminal base-pair AU7 at the bottom of the acceptor helix; it is severely broadened at 51 “C and broadens beyond detection above 55°C due to solvent exchange at rates above 200 s-l. Transfer of saturation from water to the six G.C base-pair resonances during 200 ms of water pre-irradiation (1 ms delay before the observation pulse) was carried out in a preliminary experiment (data not shown). As expected, the very slowly exchangeable aromatic C8H proton of m”G46 is relatively unaffected, whereas more than 90% of every base-pair resonance disappears when water is saturated due to base-pair exchange with the saturated water protons. This indicates that the proton exchange rate is at least 5 s-l.
184
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E.
I
HURD
I
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I
AND
I
-13
I
I
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B.
I
R.
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* -10
,I
-9
p.p.m. FIG. 2. (pH 7.0), times of resonance
Saturation recovery of the CG2 base-pair resonance at 58°C in 5 mna-sodium phosphate 100 mix-NaCl. The spectra were obtained with a 380 ps Redfield 21412 pulse after delay 1 ms, 10 ms, 20 ms, 50 ms and 100 ms following a 200 ms saturation pulse at the CG2 frequency.
Figure 2 shows the recovery from saturation of base-pair CG2 at 58°C in 5 mMsodium phosphate (pH 7-O), 100 m&r-sodium chloride. The spectra were obtained using a 200 ms pre-irradiation pulse centered at -13.17 p.p.m. to saturate the CG2 resonance, then a variable delay (1 ms to 500 ms) followed by a 0.38 ms Redfield 21612 observation pulse. Individual spectra required between five and ten minutes of signal averaging which was obtained as three separate blocks of averaged time domain data; these were Fourier transformed, phased, added, then plotted after baseline correction. Saturation recovery was also studied as a function of temperature. Figure 3 shows the data for CG3 at 52°C and 62°C. The saturation recovery rates for single resonances, such as CG3, were determined from the slope of the logarithm of fractional saturation
HELIX-COIL
DYNAMICS
IN
RNA
150 ms
I
-15
I
I
-14
I
I1
-13
I1
-12
11
-II
I
I
-10
I
I
-15
-9
I
I
I
-14
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-13
1
I
1
-12
I
1
-II
I
-10
1
1
-9
p.p.m. FIG. 3. Saturation acquired as described
recovery of the CG3 base-pair resonance at 52°C in the legend to Fig. 2 using the same sample.
and
62’C.
Spectra
were
remaining versus delay time. Semi-logarithmic plots of the saturation recovery of CG3 (-12.7 p.p.m.) at 52”C, 58°C and 62°C in 5 mM-sodium phosphate (pH 7-O), 100 mwsodium chloride are given in Figure 4. The y-intercepts of the 58°C and 62°C link are near 1.0 indicating nearly complete saturation at time zero. The approximately 0.81 y-intercept of the 52°C line does not necessarily indicate only 81 y0 saturation of
P.P.m
0.0, L
50
100 150 200 Delay time (msl
Fm. 4. The saturation recovery rate plots for CG3 at 52”C, 58°C and 62’C presented aa fractional saturation ver~u8 delay time. The slope of the natural logarithm of fraction recovered 2rewus delay time gives the following rates: 52’C (9 s-l), 58°C (18 8-l) and 62°C (35 s-l).
186
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B.
k open
R.
REID
ki [cat.]
rclose FIG. 5. A schematic diagram coil and water, axe shown.
of proton
exchange
from
the helix.
The
3 states
involved,
helix,
the CG3 resonance, however, since a small amount of a broadened base-pair from the anticodon helix is still present (albeit exchanging very rapidly) at 52°C under these solvent conditions (note the relative intensities at -12.7 p.p.m. and -12.4 p.p.m. between 51°C and 55°C compared to their relative intensities between 55°C and 61°C in Fig. 1). Between 52°C and 62°C the saturation recovery rate of the CG3 base-pair resonance increases about fourfold (from 9 s-l to 35 s - l). The relationship between logarithm rate and reciprocal temperature yields a non-linear Arrhenius plot for the three temperature points studied. The reasons for this probably lie in the dual mechanism of recovery from saturation. The two mechanisms are (1) exchange with an unsaturated water proton, and (2) the purely magnetic spin-lattice relaxation; the experimentally observed saturation recovery rate is the arithmetic sum of these two processes. The exchange process, whatever its mechanism, presumably has a specific activation energy and therefore a linear Arrhenius plot and hence we empirically sought a constant which, when subtracted from each data point, produced a linear plot. A value of about 6 s- 1 satisfied this criterion and we tentatively equate this rate as the approximate spin-lattice relaxation rate. Slthough the spin-lattice rate is not expected to be temperature-independent, it is expected to be relatively small and its temperature variation across the 10 deg. range we have studied is probably less than the combined errors in spectral peak integration and in the Arrhenius plot. Using this value together with the saturation recovery rate leads to proton exchange rates of 3 s-l at 52°C and 30 s-l at 62”C, i.e. a surprising tenfold increase in exchange rate over a 10 deg. range. The ring NH protons studied here are located in the core of the RNA helix and are inaccessible to direct exchange with water. Hence proton exchange must involve two separate processes, namely (1) a local structural opening or “breathing” followed by (2) chemical exchange from the exposed coil state. These processes are shown schematically in Figure 5. An important and interesting property to know would be the breathing or opening rate, k,,,,, for a single base-pair in a given helix. Such information for each individual base-pair would then permit a detailed analysis of the extent to which the helix-coil transition was fully co-operative throughout that helix. However, the observed proton exchange rate is not’ necessarily the helix opening rate, especially when the coil-water rate, k.[cat.], is the same or less than k open;only when k.[cat.] is very fast will the observed exchange rate be equal to, and rate-limited by, the rate of helix opening. The purely chemical coil-water rate, k.[cat.], is buffer-catalyzed and dependent upon pH, type of buffer and concentration
HELIX-COIL
DYNAMICS
IN
GCI CG4 1
RNA
187
GCI 7”
130 ms
I,
-15
I
-14
I
I
-13
,
I
-12
I
I,
-II
I
-10
I
I I I I I1
I
-15
-9
-14
-13
I I I1 -12
-II
and
CG4
I I I -10
-9
p.p.m. FIG. 6. Saturation sodium
phosphate
(pH
recovery of the 2.proton 7.0), 100 mm-NaCl.
peak
containing
GCl
at 62°C
in 5
IKIM-
of buffer (Kallenbach et al., 1976; Eigen, 1964); hence k.[cat.] can be experimentally manipulated via pH and buffer concentration and, if this step is limiting in the exchange process, such changes should dramatically affect both the exchange rate and the observed saturation recovery rate. We therefore repeated the saturation recovery experiments at various pH values, various buffer concentrations, and with additional buffers ; the results of buffer addition and pH changes under isoionic conditions are summarized in Figure 11. At 63°C and pH 5.8 the saturation recovery rate of CG3 is 13 s-l in 5 mw-sodium phosphate buffer and increases only slightly to 15 s-l in 50 mlcl-sodium phosphate buffer (total Na+ was adjusted to 408 mM in both cases by addition of N&l). The rate st pH 7.2 in 5 mM-phosphate is unaffected by the addition of 10 mnn-Trisa HCl. Lastly, instead of decreasing upon reduction of [OH-], the saturation recovery rate increases slightly at pH values below neutrality. These results conclusively demonstrate that proton exchange is not limited by the coil-water exchange rate (expect,ed to be ab least lo5 s-l in 10 rnM-Tris; see Kallenbach et al., 1976) ; hence the helix opening rate, k,,,,, must be rate-limiting in the exchangedominated saturation recovery. The small increase in saturation recovery rate as the pH is lowered must therefore reflect a slight destabilization (faster k,,,,) under acidic conditions. A similar result was observed for the adjacent base-pair CG2. Using tritium isotope methods, Teitelbaum & Englander (1975) showed that proton exchange into the ring NH of G * C helices is open-limited at low temperature ; the same appears to be true at 63°C if adequate buffer catalysis is provided to maintain rapid coil-water
188
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HURD
AND
B.
R.
REID
300 ms -
_.-_-I
a.J
160 AM CO -
---
~--
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P.P.m
(a)
I
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-12
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I
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I
.’
I
-9
(‘b)
FIG. 7. Saturation recovery of CG4 aa a single resonance at 52°C. (a) Effect of increasing Coa+ on the spectrum of E. coli tRNAPhe m 5 mmsodium phosphate (pH 7.0), 100 mu-NaCl at 62% Titration with Coa+ to 260 ELM shifts GC6 (0.26 p,p.m.), CC1 (0.17p.p.m.) and CC2 (@07p.p.m.) upfield. CC1 is shifted away from CG4 onto CC%. (b) The saturation recovery of the single CG4 resonance exposed by Co2 + .
exchange. After spin-lattice correction the saturation recovery rate can now be directly equated with k,,,,, and hence the activation energy for opening the helix at that particular base-pair can now be calculated from the temperature dependence; the activation energy for opening CG3 appears to be approximately 49 kcal/mol.
0.1 1
FICZ. 8. The semi-logarithmic data were obtained at 62°C N&l and 0.26 mM-CoCl,.
I 50
I 100
I 150
plot of the saturation on a sample containing
I 200
I 250
1 300
recovery rate of the CG4 resonance. These 6 mM-sodium phosphate (pH 7.0), 100 mM-
HELIX-COIL
DYNAMICS
IN
RNA
189
62’ C1
58O (
h
5
6
12 s-
73 s-1
I 100
1 50
I 150
I 200 T hs)
FIG. 9. Semi-logarithmic Samples
were dissolved
plots of GC5 (0) and GC6 (0) saturation recovery in 5 mm-sodium phosphate (pH 7,0), 100 nm-NaCl.
at 58°C and
62°C.
Recovery of the two unresolved resonances, GCl and CG4, at 62°C is shown in Figure 6. These data were obtained in 5 mM-sodium phosphate (pH 7.0), 100 mMsodium chloride. From simple inspection it is obvious that an intensity equivalent to one proton is rapidly recovered in the first 20 ms whereas the second proton requires about 100 ms for full recovery. The semi-logarithmic plots for this two-proton peak (not shown) revealed distinctly biphasic recovery from which the slower rate was found to be 21 s-l. Correcting for the contribution of the slower rate we determined we deduce that the slower the rate of the faster proton to be about 90 s- I. Intuitively rate is the recovery rate of the interior base-pair CG4, and that the faster rate is associated with the terminal base-pair GCl.
-15
-14
-13
-12
-II
-10
-9
-15
-14
-13
-12
-II
-10
-9
P4.m FIG. 10. Saturation phosphate
(pH
7.0),
recovery of the m’G46 100 miwNaC1.
C8H
resonance
at 58°C
and
62°C
in 5 mm-sodium
190
R.
E.
HURD
AND
Saturation
B.
R.
REID
recovery rates (s-l)
&OH
FIG. 11. Summary G *C base-pairs under
of saturation recovery a variety of conditions.
rates
determined
for
the ring
NH
protons
of the
6
Addition of Co2+ to the sample (80 PM to 260 PM) in 5 miw-sodium phosphate (pH 7-O), 100 mM-sodium chloride at 52°C shifts the terminal and penultimate basepair resonances upfield (GCl, GC6 > CG2) ; during this titration GCl is upfield shifted onto the GC5 resonance thus exposing CG4 as a single resonance. The saturation recovery of CG4 at 52°C under these conditions is shown (along with the Co2+ titration) in Figure 7. A semi-logarithmic plot of the data is shown in Figure 8 ; it is a single exponential. The rate, 6 s-l, is consistent with the other rates determined at 52°C. The exchange rate of neighboring base-pairs in a helix can be quite different as shown in Figure 9 in which the saturation recovery rates of GC5 and GC6 are plotted together at two temperatures. At both 58°C and 62”C, there is a more than fourfold difference in the recovery rates for these two adjacent base-pairs. The helix opening rate for AU7 is so rapid (>200 s- ‘) that its resonance is not even observed at 58°C (see Fig. 1). The saturation recovery rate was also determined for the non-exchangeable (at least on this time-scale) aromatic C8H of mlG46 at 58°C and 62°C and the data are shown in Figure 10. These rates, 1 s- l at 58°C and 1.4 s - l at 62”C, are presumably dominated by magnetic spin-lattice relaxation processes with very little, if any, exchange component. The increase in the spin-lattice relaxation rate from about 1 to 1.5 s-l for the largely random coil m7G46 C8H to about 6 s-l for the structured, hydrogen-bonded proton of CG3 is not unreasonable considering the increased relaxation contributed by its association with two nitrogen atoms.
HELIX-COIL
DYNAMICS
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RNA
191
4. Discussion The saturation recovery rates obtained for the six G. C base-pair resonances from the E. wli tRNAPhe acceptor stem are summarized in Figure 11. Exchange-in of saturated water protons was shown to be greater than 90% within 200 ms at 58°C. This, combined with the observation that the saturation recovery rates are highly t’emperature-dependent, indicated that the recovery process for all resonances is dominated by exchange with water protons. The buffer and pH studies clearly indicate that under these conditions the saturation recovery rates are independent of the coil-water exchange rate and reflect the different helix opening rates for individual base-pairs (combined with a much slower magnetic recovery rate). An interesting observation is that adjacent base-pairs can have quite large differences in their helix opening rates. For instance at 58°C the recovery rate of GC5 is 18 s - l (helix opening approx. 6 s- ‘) whereas GC6 recovers at 73 s - l (helix opening approx. 67 s-l) i.e. differences of approximately tenfold for nearest-neighbors at the distal end of the helix. At the open end of the helix the breathing rates (after subtracting the estimated non-exchange relaxation) are about 35 s-l for GCl and about 16 se1 for CG2 at 58°C. The small uncertainty in the precise value of the breathing rate derives from our lack of knowledge concerning the mechanism, and therefore the temperature dependence. of the non-exchange component of the relaxation. We estimate the nonexchange relaxation to have an average value of about 6 s-l across the 52°C to 62°C t,emperabure range. The classical dipolar relaxation rate for the helix will be somewhat IPSS than that of the more flexible m7G C8H (1 s-l at 58°C) and, at 360 MHz, will show a mild correlation time-dependent increase at 62’C, since the correlation time of the partially unfolded tRNA must still be well above 3 x lo-lo s. Assuming no gross structural unfolding across this temperature range, the increase in the rate of normal dipolar relaxation will be less than 20%, i.e. less than 1.2 s-l. The remaining 4 to 5 s-l of non-exchange relaxation presumably occurs via a combination of spindiffusion and quadrupolar relaxation of the ring NH protons. Relaxation via spindiffusion (Stoesz et al., 1978) is expected to decrease as the rotational correlation time is reduced by raising the temperature, and is not expected to be significant for partially unfolded molecules around 58°C (Stoesz et al. (1978) estimate this crossrelaxation rate to be about 4 s-l at 20°C for histidine NH protons in a polymer of similar size to fully structured tRNA). While spin-diffusion would complicate exchange analysis studied by transfer of saturation from pre-irradiated water to ring NH protons, it is less of a problem when the inherently rapidly relaxing ring NH protons are selectively irradiated as we have done. Hence probably the major non-exchange relaxation is via the rapid quadrupolar relaxation of the two nitrogen atoms between w,hich the ring NH proton is shared in the hydrogen-bonded state. This process is expectted to have a weak temperature dependence similar to that of dipolar relaxation. in the non-exchange relaxation These unknown effects introduce a level of uncertainty rate H hich must be subtracted from the saturation recovery rates to obtain the true breathing rates. However, a conservative estimat,e of these errors still leads to nonexchange recovery rates of 652 s- l. In the case of observed saturation recovery rates of 25 s- l or higher the error in the estimated breathing rates is less than * lo:/, which is approaching the precision of measuring peak heights at the S/N ratios in
192
R.
E. HURD
AND
B. R.
REID
our spectra. However, this error becomes more serious at slow saturation recovery rates, e.g. the rate of 9 s-l for CG3 at 52°C could correspond to breathing rates anywhere from 1 s-l to 5 s-l. For these reasons we have refrained from attempting to extract accurate activation energies for breaking each individual base-pair. There is a qualitative sequential trend to higher activation energies towards the interior of the helix (with the exception of GCl which appears to remain stacked with A73), which is consistent with obligatory dissociation of exterior base-pairs before internal ones can breathe; however, rigorous corroboration of this idea requires much more extensive data than that presented here. An obvious application of this technique is the analysis of the dynamics of each secondary and tertiary base-pair in a native tRNA molecule but there are several problems which complicate such an analysis, not the least of which is the increased ambiguity in precise assignment of overlapping resonances in crowded, complex 27-line spectra. However, the approach can be turned around and used to assist in the determination of which resonances are derived from tertiary interactions based on their kinetic behavior, as has been elegantly demonstrated by Johnston & Redfield (1977). A further complication is that processes other than exchange dominate the recovery process at lower temperatures. Johnston & Redfield (1978) have pointed out that only when saturation recovery rates become st,rongly temperature-dependent at rates above about 5 s - l can useful exchange dynamics be extracted from the data ; however, they also showed that such conditions could be met at close to physiological temperatures by appropriate manipulation of the solvent. A more conservative aim towards which we are working is the effect of permuted base sequences, including G.U pairs, on local dynamics and stability in short RNA helices such as the one described here. Such investigations are carried out at relatively high temperatures where the recovery rates are heavily dominated by exchange and under buffer conditions where exchange is open-limited. Changing the sequence of the six G * C and C. G pairs, as well as the effects of G-U or A. U replacements, will hopefully shed some light on the fundamental principles underlying the sequence dependence of the stability of RNA helices. The authors gratefully acknowledge the use of the Stanford Magnetic Resonance Laboratory (supported by National Science Foundation grant GR23633 and by National and Lillian Institutes of Health grant RR0071 1). 0 ur sincere thanks go to Susan Ribeiro McCollum for their expertise in purifying the E. coli phenylalanine tRNA used in this study. We are especially indebted to Edward Azhderian for his help in obtaining some of the spectra which were presented. We also thank Edward Azhderian and Dr Do-Lan HOO for stimulating discussions on theoretical aspects of this study. This research was SUPported by grants (to B.R.R.) from the National Science Foundation (PCM-7902904), the National Institutes of Health (GM/CA 27005) and the American Cancer Society (NP-191).
REFERENCES Arter, D. B. & Schmidt, P. G. (1976). Nucl. Acids Res. 3, 1437-1447. Eigen, M. (1964). Angew Chem. Int. Ed. England, 13, 1-19. Gillam, I., Millward, S., Blew, D., Van Tigerstrom, M., Wimmer, E. & Tener, G. M. (1967). Biochemistry, 6, 3043-3056. Holmes, W. M., Hurd, R. E., Reid, B. R., Rimerman, R. A. & Hatfield, G. W. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 1068-1071.
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Hurd, R. E. & Reid, B. R. (1979). Biochemistry, 18, 4017-4024. .Johnston, P. D. & Redfield, A. G. (1977). Nucl. Acids Res. 4, 3599-3615. Johnston, P. D. & Redfield, A. G. (1978). Nucl. Acids Re.s. 5, 3913-3927. Kallenbach, N. R., Daniel, W. E. & Kaminker, M. A. (1976). Biochemistry, 15, 121%1228. Kearns, D. R., Patel, D. & Shulman, R. G. (1971a). Nature (London), 229, 338-340. Kearns, D. R., Patel, D., Shulman, R. G. & Yamane, T. (19715). J. MoZ. Biol. 61, 265270. Nishimura, S. (1971). Procedures in Nucleic Acid Research (Cantoni, G. L. & Davies, D. R., eds), vol. 2, pp. 5422564, Harper and Row, New York. Reid, B. R. & Hurd, R. E. (1977). Act. Chem. Res. 10, 396-402. Reid, B. R., McCollum, L., Ribeiro, N. S., Abbate, J. & Hurd, R. E. (1979). Biochemistry, 18, 39964004. Stoesz, J. D.. Redfield, A. G. & Malinowski, D. (1978). FEBS Letters, 91, 320-325. Teitelbaum. H. & Enplander, S. W. (1975). J. Mol. BioZ. 92, 79-92.