High-resolution proton magnetic resonance studies of the 3′-terminal colicin fragment of 16 S ribosomal RNA from Escherichia coli

J. Mol. Biol. (1983) 170, 939-956

High-resolution Proton Magnetic Resonance Studies of the Y-Terminal Colicin Fragment of 16 S Ribosomal R N A from Escherichia coli Assignment o f Iminoproton Resonances by Nuclear Overhauser Effect Experiments and the Influence o f Adenine Dimethylation on the Hairpin Conformation H. A. HEUS, J. M. A. VhN KIMMENADE, P. H. VAN KNIPPENBERG

Department of Biochemistry, State University of Leiden Wassenaarseweg 64, 2333 A L Leiden, The Netherlands C. A. G. HAASNOOT, S. H. DE BRUI~ AND C. W. HILBERS

Department of Biophysical Chemistry, Catholic University Toernooiveld, 6525 ED Nijmegen, The Netherlands (Received 8 June 1983) The "colicin" fragments comprising the 49 3'-terminal nucleotides of 16S ribosomal RNA have been isolated from wild-type Escherichia coli and from a kasugamycin-resistant mutant that lacks methylation of two geminal adenine residues. Proton nuclear magnetic resonance (n.m.r.) spectra (500MHz) were recorded at various temperatures. The low-field resonances arising from the hydrogen-bonded iminoprotons of paired bases were assigned using the nuclear Overhauser effect (n.o.e.). Crucial to the interpretation of the spectra are the resonances that originate from the two hydrogen-bonded iminoprotons ofa U. G basepair. Combined with temperature-jump relaxation kinetics experiments the n.o.e.s lead to the conclusion that a conserved A. U / U . G junction in the hairpin is a thermolabile dislocation in the helix. The n.m.r, spectra of the wild-type and mutant fragment are only different with respect to the iminoproton resonances of the two base-pairs adjoining the hairpin loop. The spectra recorded at various temperatures tend to indicate that dimethylation of the adenosines labilizes these base-pairs, but no definitive conclusions are drawn. The results confirm our previous views that dimethylation of the adenosine residues affects the conformation of the hairpin loop.

1. Introduction Our u n d e r s t a n d i n g of the structure of the ribosome has been increased i m m e n s e l y b y the recent progress in the s t u d y of the sequence and secondary s t r u c t u r e of ribosomal R N A (Woese et al., 1980; Stiegler et al., 1981; Glotz et al., 1981), T h e presence of n u m e r o u s distinct hairpin structures, as well as certain long-range interactions via base-pairing, has been fully documented, 939 0022-2836/83/320939-18 $03.00]0

© 1983 Academic Press Inc. (London) Ltd.

940

H. A. HEUS ET AL.

The structure of ribosomal RNA is strongly conserved during evolution. In supposedly single-stranded regions one often finds strong conservation of sequence, whereas tentative double-stranded parts show many compensating base-pair changes, leaving the number of base-pairs in such stretches more or less equal. In order to provide a more detailed picture of the three-dimensional structure of ribosomal RNA, one approach would be to dissect the RNA into parts that can be handled separately and are not too large to be studied successfully by physical techniques. One such part that can be isolated relatively easily is the 3' terminus of 16S ribosomal RNA. Treatment of Escherichia coli ribosomes with the bacteriocins colicin E3 or cloacin DF13 results in specific cleavage at 49 nucleotides from the 3' end (Bowman et al., 1971; de Graaf et al., 1973). The resulting "coticin" fragment can be isolated and studied (Baan et at., 1976a,b; 1977). This part of the molecule is extremely conserved (van Charldorp & van Knippenberg, 1982) and represents a separate "domain" in secondary structure models of 16S RNA (Stiegler etal., 1981). It contains the Shine & Dalgarno (1974) sequence involved in initiation of prokaryotic protein synthesis and also two conserved N6-dimethyladenosine residues (Ehresmann et al., 1971). However, the methyl groups are lacking in mutants resistant to kasugamycin (Helser et al., 1971). A model of the secondary structure of the colicin fragment of E. coli is shown in Figure I. Previously, we have studied the structure and thermodynamic properties of the colicin fragments of wild-type and kasugamycin-resistant E. coli by a variety of physical techniques (Baan et al., 1977; van Charldorp et al., 1981a,b,1982; Heus et al., 1983). One of the conclusions emerging from these investigations was that dimethylation of the adenine bases destabilizes the secondary structure of the colicin fragment. This was a,~cribed to the strong stacking tendency of N6-dimethyladenosine.

/ G

G*--A

22

*~/ • C i

21 G -

C

20 A 19 U "

U (~

i

t

C i 17 C " G i 16 C • G i 15 A * U ! 14 h • U i m U G-U- C-G-U-A-A-C-A-A-G-G" G~G-A-U-C-A-C-C-u-C-C-U-U-A 18

5' OH

G •

5' OH

FIG. 1. Secondary s t r u c t u r e model of the coticm f r a g m e n t of E. coli. Only t h e central hairpin is shown, although the formation of additional secondary s t r u c t u r e is possible. T h e asterisks indicate N6-dimethylation of the adenosine residues in the wild-type fragment.

PROTON n.m.r. STUDIES OF THE COLICIN FRAGMENT

941

To further our understanding of the conformation of this p a r t of the ribosomal R N A the physical studies were extended using 5 0 0 M H z proton n.m.r/f spectroscopy. P r o t o n n.m.r, spectra were interpreted using nuclear Overhauser effect experiments. The n.o.e, is the change in integrated intensity of the resonance signal(s) of one set of nuclei as a result of the s a t u r a t i o n of a n o t h e r set of nuclei by a strong resonant radiofrequency field. Cross-relaxation processes in the spin system are responsible for the occurrence of these effects. On theoretical grounds one can expect effects of a b o u t 20% for spins t h a t are ~ 2.5 A a p a r t (e.g. the iminoprotons in a G • U base-pair) and of a b o u t 2 to 5O/o when the spins have a distance of 4 to 5 A (e.g. the iminoprotons in adjacent base-pairs), n.o.e. e x p e r i m e n t s have been employed successfully in the assignment of hydrogenbonded iminoproton resonances of transfer R N A s (Hare & Reid, 1982a,b; R o y & Redfield, 1981; H e e r s c h a p et al., 1982) and of D N A f r a g m e n t s (Patel et al., 1982; H a a s n o o t & Hilbers, 1983). Here we use this a p p r o a c h to assign the iminoproton resonances of the hairpin helix in the colicin f r a g m e n t of E. coli (Fig. 1). In addition, it is shown t h a t d i m e t h y l a t i o n of the adenosine residues in the loop gives rise to a conformational difference and t h a t the conserved A. U / U . G junction in the hairpin stem (van Charldorp & van K n i p p e n b e r g , 1982) represents a " w e a k " spot in the helix.

2. Materials and Methods (a) Isolation of colicin fragments The procedure for the isolation of the 49 nucleotide fragment from E. coli was basically the same as that described by Baan et al. (1976a). All glassware, plastics, buffers etc. were sterilized before use. In a routine isolation procedure 50,000A260 units of high-saltwashed ribosomes obtained from 200 g of frozen E. coli cells were treated with 1 mg of the bacteriocin cloacin DF13 (which acts exactly as colicin E3; de Graaf et al., 1973). The extent of 16 S RNA cleavage can be estimated by gel electrophoresis as described by Baan etal. (1976b). The treated ribosomes were dissociated by adjusting the magnesium concentration to 1 mM and the subunits were separated by sucrose gradient centrifugation in a zonal rotor. Under these conditions the colicin fragment stayed with the 30 S particles. The RNA was extracted with phenol and fractionated using sucrose gradients in swing-out rotors. A small hump in the A 260 profile near the top of such gradients signals the presence of the fragment. The corresponding fractions were pooled, the RNA was precipitated and collected by centrifugation. The material was not pure fragment as judged by gel electrophoresis and had to be passed over a Sephadex G150 column, which separates the colicin fragment from other contaminating RNA species. The fractions containing the fragment in water were freeze-dried. A typical yield was about 80A26 o units (3"5 mg) of fragment from 50,000 A260 units of ribosomes. Figure 2 shows the optical density scans after gel electrophoresis of isolates from wildtype E. coli and its kasugamycin-resistant descendant. Previously (Baan et al., 1976a), the possibility of the presence of smaller oligonucleotides was determined by high-pressure liquid chromatography. Another way of checking for the presence of contamination that we used was 5'-end-labelling with 32p by incubation with [~-32P]ATP and polynucleotide kinase, followed by polyacrylamide gel electrophoresis and autoradiography. This worked well for contamination with small oligonucleotides, but we had several preparations that t Abbreviations used: n.m.r., nuclear magnetic resonance; n.o.e., nuclear Overhauser effect; p.p.m., parts per million.

942

H. A. HEUS E T A L . ]/

x

B

x

e

Migration

Fro. 2. Ultraviolet scans of polyacrylamide gels of purified colicin fragments. I, Wild-type fragment; II, mutant fragment; X, xylene cyanol FF marker; B, bromophenol blue marker.

showed only one band by this technique, but gave several ultraviolet light-absorbing bands in cylindrical gels due to the presence of very large fragments of ribosomal RNA. All preparations were therefore purified until they gave a pattern as shown in Fig. 2. Most of the preparations used were checked by sequencing methods to determine their identity. The wild-type or m u t a n t character of the fragment is betrayed by a characteristic difference in mobility in gel electrophoresis (van Charldorp et al., 1981b) and can be further easily demonstrated because of the resistance of the m~A-m~A sequence to ribonuclease U2 and chemical modification. The final buffer conditions for the n.m.r, experiments were attained by dissolving the freeze-dried BNA directly in 0.2 ml buffer (12.5 mM-NaH2PO 4, 12.5 mM-Na2HPO 4, 1 raMsodium cacodylate, 61.5 mM-NaCl, l mM-EDTA (pH 7) containing 5% 2H20 ). The amount of RNA varied between 2 and 4 mg per 0"3 ml of buffer. (b) n.m.r, experiments IH n.m.r, spectra (500 MHz) were recorded on a Bruker WM500 spectrometer equipped with an Aspect 2000 minicomputer. The spectrometer was operated in the Fourier transform mode using quadrature detection. Chemical shifts are quoted relative to DSS (2,2-dimethyl-2-silapentane-5-sulphonate). Downfield shifts are defined as positive. Chemical shifts were measured relative to the solvent water peak and converted to the DSS reference by temperature and salt calibration curves. Excitation of the water peak was reduced by using a combination of a semi-selective "Redfield 214 pulse" (Redfield & Kunz, 1979) and so-called alternate delayed acquisition (Roth et al., 1980). We have used this approach successfully in the study of a t R N A (Heerschap et al., 1982) and a DNA fragment (Haasnoot & Hilbers, 1983). The Bedfield 214 pulse had a total length of 250 ps; the carrier was offset 4000 Hz from the water resonance; the acquisition time was 0"82 s and the relaxation delay 0"3 s. No baseline corrections were made. Nuclear Overhauser effect experiments were recorded using the same technique; however, in these experiments, a preirradiation pulse was given prior to the semi-selective observation pulse set on-resonance of one of the iminoprotons. Subtraction from a spectrum with an offresonance preirradiation pulse yields the desired n.o.e, difference spectrum. To perform the proper experiments the preirradiation pulse has to be of sufficiently low power (35 dB below 0-1 W) to achieve selective saturation of the resonance at a position A and short

PROTON

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OF THE COLICIN FRAGMENT

943

enough to avoid extensive spin diffusion effects outside the sphere of the nearest neighbours of A. In our case a preirradiation time of 0.3 to 0.4 s was used. n.o.e, difference spectra were given a line-broadening of 5 to l0 Hz for a better signal-to-noise ratio. (c) Temperature-jump and optical melting experiments The measurements were carried out on a double-beam temperature-jump instrument, equipped with a modified version ofa Messanlage T-jump cell with a sample volume of 1-0 ml. The cell temperature was varied using a cryothermostat (Metter, WKS) and by a copperconstantan thermocouple connected to a high-resolution digital voltmeter (Solartron LM 1440.3). Heating pulses were given that resulted in temperature jumps of 1-5 to 2-5 deg. C. A Zeiss (MQ3) monochromator was employed; the light source was a diffused mercury-xenon arc lamp (Conrad-Hanovia, L5005-I00). To avoid lamp ripple the beam was split. The outputs of the reference and sample photomultipliers (RCA IP28VI) were fed to a low-noise differential amplifier. In most cases we used only 4 or 5 dynodes on the photomultiplier. The photomultipliers were operated with 2 high-voltage power supplies (VG electronics). Rapid changes in transmission caused by the temperature jump were recorded using 2 transient recorders (Biomation model 805). The output of the transient recorders were observed on an oscilloscope (Philips PM3211). The data were subsequently processed in a PDP 31/24 computer, interfaced to the temperature-jump apparatus. Buffer conditions were the same as in the n.m,r, experiments. Optical melting experiments were performed as described (van Charldorp et al., 1981a).

3. Results (a) Assignment of the iminoproton resonances The low-field region of a 500 MHz proton n.m.r, spectrum of the wild-type fragment at room temperature is shown in Figure 3. Crucial to the interpretation of this spectrum are peaks 8 and 9 at l l.5 and 10.7 p.p.m., respectively, which originate from the two iminoprotons of the base-pair 19 U . G (Fig. 1). I n transfer R N A also the two carbonyl-oxygen-bonded iminoprotons of G . U pairs are clearly

9 2

i

14

i

13

r

12

i

II

I0

p,p.m,

FIo. 3. Spectrum (500 MHz) n.m.r, of the wild-type colicin fragment at room temperature. See Materials and Methods for experimental details.

944

H. A. H E U S E T A L .

distinguishable from the ring-nitrogen-bonded iminoprotons of regular WatsonCrick base-pairs (Johnston & Redfield, 1978). Calculations based on the crystal structure of yeast tRNA Phe (Kim, 1976), which contains a U. G pair in the amino acceptor stem, predict a distance between the iminoprotons of this basepair of approximately 2.5 A. Hence, a n.o.e, of about 20~o should be measured when the sample is preirradiated at either peak 8 or peak 9, provided that the rotational correlation time, vc, is long enough. This is found indeed as shown in Figure 4: saturation of peak 8 by a preirradiation pulse gives rise to a n.o.e, on peak 9 of about 20~o and vice versa. Saturation of peak 9 results in a n.o.e, of similar magnitude on peak 8. In addition, we see a n.o.e, of smaller magnitude at the position of resonance 5 at 12.8 p.p.m. Recent work on transfer RNA has shown that n.o.e.s between iminoprotons of adjacent base-pairs are of the order of 5% (Roy & Redfield, 1981; Roy et al., 1982; Hare & Reid, 1982a,b; Heerschap et al., 1982). Peak 5 therefore originates from a base-pair adjoining 19 U.G. Its position (about 12-8 p.p.m.) suggests that it is a G. C pair and since we have other evidence that peak 2 corresponds to 20 A. U (compare below) we assign peak 5 to 18 G. C. Possible reasons why we do not observe a n.o.e, from 19 U. G on 20 A. U will be discussed below. A compilation of all the n.o.e, data is given in Figure 5. Saturation of resonance 5 gives, in addition to the expected n.o.e.s on peaks 8 and 9, a n.o.e, on peak 3, which is therefore assigned to 17 C.G. Saturation of peak 3 gives the expected reverse n.o.e, on peak 5 and, in addition, a n.o.e, on peak 7. The latter resonance is therefore assigned to 16 C. G. Except for the reverse n.o.e, on peak 3, saturation of peak 7 produces no other detectable effects. This would be

15

14

13 p.p.m,

12

II

15

14

13 p.p,m.

12

II

Fro. 4. Example of a nuclear Overhauser effect. The lower trace presents the low-field spectrum of the mutant fragment at room temperature. The upper trace gives the computer-calculated n.o.e. difference spectrum after preirradiation of peak 8 (left) and peak 9 (right).

P R O T O N n.m.r. S T U D I E S OF T H E C O L I C I N F R A G M E N T •

Y V

G

•

\ •_

1 rn2~ m2G~"G • C/ 4

....

•

V

v

•

V

v

v

•

v

, V

V

•

5

945

v

• C 6 A. U 2 U o G 8/9 5 G*C C.G C,G

3 7

A,U A,U

l?

FIO, 5. Summary of n.o.e, data. ( 0 ) Saturation of a resonance. When the difference spectrum gave a peak above background this is indicated by V at the appropriate resonance. The hairpin structure at the right shows the proposed assignments.

understandable if our previous assignment t h a t 15 A . U gives rise to the broad peak 1 (Baan et al., 1977) is correct. We are now left with three unassigned resonances, peaks 2, 4 and 6. Since peak 2 is closest to the intrinsic line position of an A . U pair (14.5p.p.m.; Shulman et al., 1973) it is most likely t h a t this resonance originates from 20 A . U . Experimental evidence indicating t h a t peak 2 is indeed due to an A- U pair is shown in Figure 6. Preirradiation of resonance 2 gives rise to a sharp n.o.e, a t 6-9 p.p.m., in addition to a n.o.e, on peak 6. Such a n.o.e, is typical for a standard A- U pair; the resonance at 6.9 p.p.m., which is due to the aromatic C-2 proton of the adenine ring, is affected by the saturation of the nearby ring N-3H of uracil (Sanchez et al., 1980). G . C pairs give rise to much broader n.o.e.s in this spectral region due to transfer of magnetization to aminoproton resonances. The n.o.e, on peak 6, observed after preirradiation of resonance 2 assigns this peak to 21 G-C. However, we could not detect a reverse

l~p.m. Fro. 6, n,o,e, difference spectrum i n c l u d i n g the aromatic p r o t o n region (7 to 9 p.p,m.) obtained after

preirradiation at the frequency of resonance 2.

946

H. A. H E U S

ET AL.

n.o.e, on peak 2 after saturation of peak 6, and neither could we find an effect on the last unassigned resonance 4, which we take to originate from 22 G . C . Although Figure 5 shows a s u m m a r y of n.o.e, d a t a of the wild-type fragment, the results with the RNA of the m u t a n t strain are in complete agreement. (b) Effects of adenosine dimethylation on the n.m.r, spectra Our previous studies have shown t h a t methyl groups on the adenine bases in the loop (Fig. l) destabilize the RNA helix (van Charldorp et al., 1981a; Heus et al., 1983). The melting temperature of the double-stranded region is (at 0-015 MNa +) approximately 5 deg. C higher in the fragment from the m u t a n t strain t h a t lacks these methyl groups. I t was therefore of considerable interest to investigate the effects of methylation in detail by proton n.m.r, at various temperatures. Figure 7 illustrates the 500 MHz spectra of wild-type and m u t a n t fragments at room temperature. On visual inspection two differences are easily observed: resonance 4, assigned above to base-pair 22 G . C is shifted downfield; and resonance 6, assigned to base-pair 21 G . C , is shifted slightly upfield for the m u t a n t fragment. Although we cannot at the m o m e n t rationalize the direction and the magnitude of these chemical shifts, they signal a conformational difference in the loop region of the molecule.

m

G'm6~'~/m6 ." 2 I

G.~

•

C

GoC

4

6

A" U 2 U • G 8/9 G. C 5 I ¢.G

3

A • U I? h" U

1'5

5I 16

I

14

3

n i 4lll

~

.

; =

A II

13 p.p.rn.

12

a I

9

I

i

Wild-'type

II

Fro. 7. The 500 MHz spectrum of wild-type (upper trace) and mutant fragments (lower trace). Assignments from n.o.e.s are shown in the hairpin structure at the left. Arrows indicate peaks with a chemical shift different from those of the wild-type fragment.

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STUDIES

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Figures 8 and 9 show the 500 MHz spectra of wild-type and mutant fragments at temperatures varying from - 3 ° C to 64°C. Resonance 1, which we ascribed to the base-pair 15 A-U, increases in intensity when the temperature is lowered, probably indicating an increasing contribution from the terminal base-pair 14 A-U. Upfield, additional resonances that we cannot clearly distinguish appear at lower temperatures. A salient feature of the spectra at low temperatures is the behaviour of the resonance that would be most influenced by a difference in loop conformation, i.e. resonance 4 originating from the top base-pair 22 G-C. On lowering the temperature this resonance stays in the wild-type spectra, but disappears in the mutant spectra. Although several additional resonances appear at low temperature, we were not able to find one that could account for the vanishing peak 4. Above 30°C peak 1 (15 A. U) disappears from the spectra. Peaks 2 (20 A. U) and 4 (22 G-C) start to shift above 24°C. Peak 2 is clearly broadening ahead of

5

7

s

9

25 °C

2÷5,67

8

9

17oC ~

1

~

I

7

°

C

12°C

~

7oC

~

15

14

ZoC

5

7

15 12 p.p.m, (o)

I

°

C

~ 5

°C

-5°C

8

II

-I °C

15

14

13 12 p.p.m. [b)

II

Fro. 8. (a) Spectra (500 MHz) of wild-type fragment at temperatures from 27°C to -3°C. (b) Spectra (500 MHz) of mutant fragment at temperatures from 17°C to - ]°C. Note (arrow) the disappearance of peak 4 below II°C.

948

H. A. H E U S E T A L .

64oC 60°C ~

56°C

"~i

40°C

2

I'5

14

8

13 p,p,m. (a)

;'2

l'l

F*o. 9. (a) Spectra (500MHz) of wild-type fragment at temperatures from 24°Cto 64°0, (b) Spectra (500 MHz) of mutant fragment at temperatures from 24°C to resonance 4 at elevated temperatures.

64°C.Arrows indicate the position of

P R O T O N n.m.r, S T U D I E S OF T H E COLICIN F R A G M E N T

64oC 60°C

56°C

"~'-=~-~

48oc

~

4

2

3

,'s

,4

5

A s

°C

8 7

,'s p,p.m, Fro.9.

A

,'2

9 '

,',

949

950

H. A. H E U S E T A L .

the other resonances and probably this is also the case for peak 4. The shift of peak 4 might bring this resonance underneath peak 5 in the wild-type spectrum and this makes it impossible to conclude, as these spectra might suggest at first glance, that the resonance due to 22 G. C in the wild-type fragment disappears at lower temperatures than in the mutant fragment. Similarly, shifting and broadening of peak 6 (21 G .C) makes it hazardous to draw conclusions on differences in the stability of this base-pair between wild-type and mutant. Nevertheless, there is evidence suggesting that in these spectra the differences in thermal stability of the resonances between mutant and wild-type is limited to the two base-pairs adjacent to the loop. One should realize that the melting temperatures of the two helices differ by only about 3deg. C under these salt conditions (van Charldorp et al., 1981a; Heus et al., 1983). At around 60°C the sharpest resonances in the spectra and the last ones to disappear are peaks 3, 5 and 7, originating from 17 C'G, 18 G . C and 16 C-G, respectively. They form the nucleation centre of the helix. The fact that peak 2 (20 A-U) broadens and disappears before peak 6 (21 G" C) suggests that melting of the helix is not only initiated at the ends, but also at the A. U/U. G junction. That this junction presents a "weak" spot in the helix follows from the experiments described in the next section. (c) Optical melting and temperature-jump relaxation experiments Line broadening in n.m.r., as shown as a function of temperature for 20 A. U and 17 C-G in Figure 10, is not necessarily a measure for the lifetime of the double helix. Therefore, to be able to combine the present results with the equilibrium melting behaviour of the double helix also, optical melting and temperature-jump experiments were carried out on the samples. An example of a differential melting curve obtained in an equilibrium melting

17C.G 80

Z°A°U

,~

N :E

40

o

,

0

.

20

,

,

40

,

.

60

,

80

t (°C) FIO. I0. Line-widths a t half-height (v½) as a function of temperature (t) for 20 A . U and 17 C.G. (O) Values calculated for wild-type fragment; (A) values calculated for m u t a n t fragment.

PROTON n.m.r. STUDIES OF THE COLICIN FRAGMENT

951

experiment is shown for the mutant fragment at 200 mM-Na + in Figure 11. Only one transition is observed with a melting temperature of 76°C and a half-width corresponding to 80 kcal/mol (1 cal --- 4.184 J). Concurrent with this observation, in the temperature-jump experiments we found only one relaxation time corresponding with helix formation throughout the whole range of temperatures studied (4°C to 84°C). The relaxation times measured for the wild-type as well as the mutant fragment are presented in Figure 12 as a function of the reciprocal absolute temperature. Within experimental accuracy the results obtained for the two fragments are equal. For the intramolecular helix-to-coil transition the relaxation time T and the formation and dissociation rate constants are related by: z -1 = K f - { - K d. At low temperature z- 1 is determined by the rate of helix formation Kf. As can be seen in Figure 12, T tends to level off to ~,-2 x 10-4s below 64°C, corresponding to Kf---0-5x 104s -1. At high temperature z -1 is determined by the rate of dissociation Kd, so in this region z equals the double helix lifetime 1 / K d. Under suitable buffer conditions, every time the helix opens up, ring iminoprotons exchange with water. In this situation line-broadening and helix lifetime are related by (Hilbers, 1979): 1 / K d = 7zAv, where

Av = v ½ - v o.

vo represents the line-width at half-height in the absence of line-broadening; v½ is the observed line-width. The 1 / K d values calculated from the line-broadening of 17 C . G and 20 A-U are also plotted in Figure 12. It is seen that for 17 C-G, one of the base-pairs of the nucleation centre, the helix lifetime determined by n.m.r. at low temperatures can be nicely extrapolated to the measured helix lifetime at high temperature. The slope of the line corresponds to a dissoeiation activation energy of 73kcal/mol (1 e a l = 4 . 1 8 4 J ) in reasonable agreement with the dissociation enthalpy obtained from the equilibrium melting experiments. Hence,

12

,~ O

8

x k <~

o o

'

4'o

'

6'o

'

'

(°C) Fie. 11. Differential ultraviolet melting curve (AA/AT v e r ~ t) of m u t a n t fragment at 200 mM-Na +.

H. A. HEUS ET AL.

952

t(oC) 50 I0-I

40

50

60

70

80

I

!

I

I

I

IO-Z

iO-~

I0-4

10-5 3-3

3.2

3.!

3.0

2-9

2-8

I / T x I 0 ~ {K -l)

FIo. 12. Relaxation time Tas a function of reciprocal temperature of (0) wild-type and (A) mutant fragments at 100 mM-Na+, Ka values calculated from n.m.r, data for the 20 A. U and 17 C. G basepairs are also indicated.

the line-broadening of the pairs 16, 17 and 18 is determined by the lifetime of the helix. This is clearly not so for the iminoprotons of the base-pair 20 A - U (and 19 G - U , not shown). The iminoprotons of these base-pairs start to exchange at lower temperature indicating t h a t they form a weak spot in the double helix. 4. Discussion (a) Assignment of iminoproton resonances by n.o.e, experiments The most successful strategy in assigning iminoproton resonances from nucleic acid double helices is to start with a solidly assigned resonance and then to look for n.o.e.s to neighbouring iminoprotons. Subsequently, the newly assigned resonances are used as further starting points. Following such a step-by-step procedure, ideally, an unambiguous assignment of the spectrum can be obtained. For the molecules studied in this paper the resonances of the U . G pair can reliably serve as such a starting point. The step-by-step procedure, however, turned out not to be entirely applicable. For the base-pairs near the ends of the helix, especially 15 A. U and 14 A. U, of which only the former gives rise to a broad resonance, the lack of a n.o.e, probably reflects the rapid exchange of the iminoprotons with water. Complete assignment by n.o.e, is further hampered b y the peculiar behaviour of base-pair 20 A. U. There is no n.o.e, of either one of the 19 U . G iminoprotons on the iminoproton of 20 A. U and vice versa. Although

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saturation of the 20 A. U resonance gives a n.o.e, on the neighbouring pair 21 G- C (peak 6), the reverse is not true. In spite of these problems the combination of n.o.e.s, melting characteristics and differences in the spectra between the mutant and wild-type fragment make the assignment as shown in the inset of Figure 5 self-consistent and inevitable. The peculiar behaviour of the 20 A. U pair deserves some special attention. The fact that no n.o.e, on the iminoproton resonance of this base-pair is observed either after saturation of peaks 8 or 9 (19 U. G), or peak 6 (21 G-C), could be explained by the assumption that this proton is subject to an exchange reaction with water. Peak 2, which is ascribed to this base-pair, broadens and disappears from the spectrum at lower temperatures than do the other resonances (excluding the base-pairs at the ends of the helix) and this supports the idea that an exchange reaction is causing the lack of a n.o.e. However, the data show an additional peculiarity. Whereas saturation of peak 2 (20 A • U) exerts a clear n.o.e. on peak 6 (21 G" C), there is absolutely no effect on peaks 8 and 9 of the other neighbour, 19 U-G. This suggests that the iminoproton of 20 A . U is further removed from the iminoprotons of the 19 U. G pair than from that of the 21 G. C pair. This is in line with a proposal by Mizuno & Sundaralingam (1978) that an U-G pair has more overlap with the base-pair at the 3' side of the G residue (and hence at the 5' side of U residue) than with the base-pair at the other side. The universal A. U / U . G junction in this helix (van Charldorp & van Knippenberg, 1982) probably presents a dislocation or a weak site in the helix. Melting of the helix starts at both ends and at this junction. (b) Conformational effects of adenine dimethylation On the basis of our previous results we have attributed the difference in helix stability between the mutant and wild-type colicin fragments to the enhanced stacking interactions of the dimethyl adenines in the loop of the wild-type molecule (van Charldorp et al., 1981a,1982; Heus et al., 1983). Using 13C n.m.r, on a coticin fragment that was specifically enriched with ~3C in the methyl groups we found that the dimethyl adenine residues are indeed strongly stacked as in the dinucleotide m~A-m~A (van Charldorp et al., 1982). We reasoned that the strong stacking tendency of the dimethylated adenines exerts a conformational strain on the hairpin structure leading to an overall destabilization. The proton n.m.r. experiments reported here reveal that methylation affects the line positions of the iminoproton resonances of the base-pairs near the loop. Although the difference in chemical shifts of these resonances between wild-type and mutant cannot yet be interpreted in terms of loop conformation, they indicate clear structural differences. The kinetic properties of the wild-type and mutant fragments are the same within experimental accuracy. This obtains for the overall kinetic behaviour as determined from the temperature-jump experiment as well as for the kinetic behaviour of the individual base-pairs, as seen by n.m.r. (cf. Figs 10 to 12). Recently, we became aware of an additional factor that might be relevant to differences in the loop conformation between the wild-type and mutant colicin

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fragments. It was pointed out by Traub & Sussman (1982) that many hairpins in ribosomal RNA, either at the loop end or at the "open" end are closed by juxtaposition of a G with an A residue. In the crystal structure of yeast tRNA such a hydrogen-bonded pair is indeed found at the open end of the anticodon helix (Kim, 1976). If it occurred at the "looped" end of the helix, it would in our case mean the closing of a loop by two nucleotides. Sterically this is not impossible and in fact such a construction was proposed for the variable loop of E. coli tRNA s~r (Brennan & Sundaralingam, 1976). Dimethytation of the two adenine residues, which makes the N 6 positions unavailable for hydrogen-bonding, clearly interferes with the possibility of closing this hairpin with G. A and leads to an alternative loop structure in which the two (dimethylated) adenine residues occupy the preferred stacked structures. (Note that monomethylation of G23 at the N ~ position, which occurs in the wild-type and mutant fragments (van Charldorp et al., 1981c), does not interfere with the base-pairing possibilities of this nucleotide). Our n,m.r, experiments, however, do not support the presence of a G. A pair in the mutant fragment. In contrast to the situation with tRNA P~e, where the presence of a G . A pair on top of the anticodon stem was unambiguously identified by iminoproton n.m.r. (Heerschap et al., 1982; Johnston & Redfield, 1981), we cannot assign a resonance to such a pair in the fragment of the mutant strain. On the other hand, because of possible fraying events, such a resonance might be obliterated and it cannot be ruled out entirely. (c) Biological implications Nature has made a tremendous effort to preserve an enzyme system in almost all organisms (van Charldorp & van Knippenberg, 1982) that is specific for the methylation of these adenosine residues. Nevertheless, kasugamycin-resistant mutants of E. coli (Helser et al., 1971) and Bacillus stearothermophilus (van Buul et al., 1983) as well as yeast mitochondrial ribosomes (Klootwijk et al., 1975; N. Martin, personal communication) are able to live without this modification. Also, in vitro it is very difficult to find an effect on the function of the ribosome. Except for the fact that dimethylation makes the particles sensitive to the antibiotic (Poldermans et al., 1979a), only very small differences between wildtype and mutant ribosomes, in the absence of the antibiotic, have been found in the requirements for initiation of protein biosynthesis and in subunit association (Poldermans et al., 1979b,1980). Possibly, the conserved sequence in the loop G-m~A-m26A and the conserved stem sequence A - U / U . G are part of a site that has to be opened transiently during the functioning of the ribosome, and methylation of the adenosines facilitates this. Detailed models for the unfolding of this hairpin for initiation and for subunit interaction have been proposed by others (van Duin et al., 1976; Azad, 1979). The n.m.r, experiments were performed on the 500 MHz facility of the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) in Nijmegen. We thank P. van Dael for keeping the instrument in excellent condition. We also thank R. van Charldorp, J. Joordens and J. H. M. van Delft for important contributions.

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C.A.G.N. and J.M.A.v.K. are supported by grants from the Netherlands Foundation for Chemical Research (SON). REFERENCES Azad, A. A. (1979). Nucl. Acids Res. 7, 1913-1929. Baan, R. A., van Charldorp, R., van Leerdam, E., van Knippenberg, P. H., Bosch, L., de Rooy, J. F. M. & van Boom, J. H. (1976a). F E B S Letters, 71, 351-355. Baan, R. A., Duijfjes, J. J., van Leerdam, E., van Knippenberg, P. H. & Bosch, L. (1976b). Proc. Nat. Acad. Sci., U.S.A. 73, 702-706. Baan, R. A., Hilbers, C. W., van Charldorp, R., van Leerdam, E., van Knippenberg, P. H. & Bosch, L. (1977). Proc. Nat. Acad. Sci., U.S.A. 74, 1028-1031. Bowman, C. M., Sidikaro, J. & Nomura, M. (1971). Nature New Biol. 234, 133-137. Brennan, T. & Sundaralingam, M. (1976). Nucl. Acids Res. 3, 3235-3251. de Graaf, F. K., Niekus, H. G. D. & Klootwijk, J. (1973). F E B S Letters, 35, 161-165. Ehresmann, C., Fellner, P. & Ebel, J.-P. (1971). F E B S Letters, 13,325-328. Glotz, C., Zwieb, C., Brimacombe, R., Edwards, K. & Kossel, H. (1981). Nucl. Acids Res. 9, 3287-3306. Haasnoot, C. A. G. & Hilbers, C. W. (1983). Biopolymers, 22, 1259-1266. Hare, D. R. & Reid, B. R. (1982a). Biochemistry, 21, 1835-1842. Hare, D. R. & Reid, B. R. (1982b). Biochemistry, 21, 5129-5135. Heerschap, A., Haasnoot, C. A. G. & Hilbers, C. W. (1982). Nucl. Acids Res. 10, 6981-7000. Helser, T. L., Davies, J. E. & Dahlberg, J. E. (1971). Nature, New Biol. 235, 6-9. Heus, H. A., van Kimmenade, J. M. A., van Knippenberg, P. H. & Hinz, H.-J. (1983). Nncl. Acids Res. 11,203-210. Hilbers, C. W. (1979). In Magnetic Resonance Studies in Biology (Shulman, R. G., ed. ), pp. 1-43, Academic Press, New York. Johnston, P. D. & Redfield, A. G. (1978). Nucl. Acids Res. 5, 3913-3927. Johnston, P. D. & Redfield, A. G. (1981). Biochemistry, 20, 1147-1156. Kim, S. H. (1976). Prog. Nucl. Acids Res. Mol. Biol. 17, 181-216. Klootwijk, J., Klein, I. & Grivell, L. A. (1975). J. Mol. Biol. 97, 337-350. Mizuno, M. & Sundaralingam, M. (1978). Nncl. Acids Res. 5, 44514461. Patel, D., Koztowsky, S. N., Kraky, L. A., Broka, C., Rice, J. H., Itakura, K. & Breslauer, K. J. (1982). Biochemistry, 21,428-436. Poldermans, B., Goosen, N. & van Knippenberg, P. H. (1979a). J. Biol. Chem. 254, 90859089. Poldermans, B., van Buul, C. P. J. J. & van Knippenberg, P. H. (1979b). J. Biol. Chem. 254, 9090-9093. Poldermans, B., Bakker, H. & van Knippenberg, P. H. (1980). Nucl. Acids Res. 8, 143-151. Redfield, A. G. & Kunz, S. D. (1979). In N M R and Biochemistry (Opalla, S. J. & Lu, P., eds), pp. 225, Marcel Dekker, New York. Roth, K., Kimber, B. J. & Feeney, J. (1980). J. Magn. Res. 41, 302-309. Roy, S. & Redfield, A. G. (1981). Nucl. Acids Res. 9, 7073-7083. Roy, S., Papastravos, M. Z. & Redfield, A. G. (1982). Biochemistry, 21, 6081-6088. Sanchez, V., Redfield, A. G., Johnston, P. D. & Tropp, J. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 5659-5662. Shine, J. & Dalgarno, L. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 1342-1346. Shulman, R. G., Hilbers, C. W., Kearns, D. R., Reid, B. R. & Wong, Y. P. (1973). J. Mol. Biol. 78, 57-69. Stiegler, P., Carbon, Ph., Ebel, J.-P. & Ehresmann, C. (1981). Eur. J. Biochem. 120, 487495. Traub, W. & Sussman, J. L. (1982). Nucl. Acids Res. 10, 2701-2708. van Buul C,P. J . J , Datum, J. B. L. & van Knippenberg P.H. (1983). Mol: Gen. Genet. 189,475478. van Charldorp, R. & van Knippenberg, P. H. (1982). Nncl. Acids Res. 10, 1149-1158.

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van Charldorp, R., Heus, H. A., van Knippenberg, P. H., Joordens, J., de Bruin, S. H., & Hilbers, C. W. (1981a). Nucl. Acids Res. 9, 4413-4422. van Charldorp, R., Heus, H. A. & van Knippenberg, P. H. (1981b). Nucl. Acids Res. 9, 267-275. van Charldorp, R., Heus, H. A. & van Knippenberg, P. H. (1981c). Nucl. Acids Res. 9, 2717-2725. van Charldorp, R., Verhoeven, J. J., van Knippenberg, P. H., Haasnoot, C. A. G. & Hilbers, C. W. (1982). Nucl. Acids Res. 10, 4237-4245. van Duin, J., Kurland, C. G., Dondon, J., Grunberg-Manago, M., Branlant, C. & Ebel, J.-P. (1976). F E B S Letters, 52, l l l - l l 4 . Woese, C. R., Magrum, L. J., Gupta, R., Siegel, R. B., Stahl, D. A., Kop, J., Crawford, N., Brosius, J., Gutell, R., Hogan, J. J. & Noller, H. F. (1980). Nucl. Acids Res. 8, 22752293. Edited by M. Gellert