Nuclear magnetic resonance studies of codon-anticodon interaction in tRNAPhe

J. Mol. Biol. (1980) 142, 195-217

Nuclear Magnetic Resonance Studies of Codon-Anticodon Interaction in tRNAPhe I. Effect of Binding Complementary Tetra and Pentanucleotides to the Anticodon H. A. M. GEERDES~,

J. H. VAN BOOMS AND C. W. HILBERS~

‘Department of Biophysical Chemistry University of Nijmegen Toernooiveld 6525 ED Nijmegen The Netherlands 2Gorkzeus

Laboratoria

State University of Leiden P.O. Box 9502 2300 AA Leiden The Netherlands (Received 10 January

1980, and in revised form 28 May 1980)

The effect of codon-anticodon interaction on the structure of two tRNAPhe species was investigated by means of nuclear magnetic resonance spectroscopy. To this end n.m.r.t spectra of yeast and Escherichiu coli tRNAPhe were recorded in the absence and the presence of the oligonucleotides U-U-C-A, U-U-C-G and U-U-C-A-G, which all contain the sequence WC complementary to the anticodon sequence GAA. The spectra of the hydrogen-bonded protons, the methyl protons and the internucleotide phosphorous nuclei served to monitor the structure of the anticodon loop and of the tRNA in the tRNA-oligonucleotide complex. From the changes in the methyl proton spectra and in the phosphorous spectra it could be concluded that the oligonucleotides bind to the anticodon. Moreover it turned out that the binding constants obtained from these n.m.r. experiments were, within experimental error, equal to the values obtained with other techniques. Using the resonances of the protons hydrogen-bonded between the oligonucleotide and the anticodon loop the structure of the latter could be studied. In particular, binding of the pentanucleotide U-U-C-A-G, which is complementary to the five bases on the 5’ side of the anticodon loop, resulted in the resolution of four to five extra proton resonances indicating that four t,o five base-pairs are formed between the pentanucleotide and the anticodon loop. The formation of five base-pairs was confirmed by an independent fluorescence bindihg study. The resonance positions of the hydrogen-bonded protons indicate, that an RNA double helix is formed by the anticodon loop and U-U-C-A-G with the five base-pairs forming a continuous stack. This structure can be accomodated t Abbrevietions

used: n.m.r., nuclear magnetic

resonance;

p.p.m., pitrts per million.

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in the so-called 5’ stacked conformation of the anticodon loop, E structure that has been suggested earlier as an alternative to the familiar 3’ stacked conformation in the crystal structure models of yeast tRNAPhe. It turned out that structural adjustments of the ant,icodon loop t,o the binding of the oligonucleotides are propagated into the anticodon stem. The relevance of these results with respect to the mechanism of protein synthesis is discussed.

1. Introduction One of the most important steps during protein synthesis is the recognition between the triplet codon on the messenger RNA and the anticodon of the corresponding transfer RNA. This recognition step is of a remarkable specificity, which is a prerequisite for the production of proteins with the proper sequence of amino acids. This behaviour does not have its counterpart in the association reactions of model systems. Physico-chemical studies of base-pairing in short oligonucleotides have demonstrated that the association of complementary trinucleotides in dilute solutions is virtually undetectable (Jaskunas et al., 1968). ,4s a consequence one might simply attribute the specific interaction between tRNA and mRNA to some property of the ribosome. A number of recent experiments have demonstrated that this proposition cannot be entirely correct. It turns out that the binding constants between anticodon loops in tRNAs and complementary oligonucleotides are much higher than between the corresponding complementary oligonucleotides, indicating that special structural features of the tRNA molecules contribute to this enhanced affinity (Uhlenbeck et al., 1970; HGgenauer, 1970; Uhlenbeck, 1972; Hiigenauer et al., 1972; Eisinger et al., 1970; Pongs et al., 1973; Eisinger & Gross, 1975; Grosjean et al., 1976; Yoon et aZ., 1975). The three-dimensional structure of yeast tRNA Phe derived from single-crystal X-ray diffraction experiments provides a firm basis for the study of structurefunction relationships in tRNA. Already, before the X-ray model of yeast tRNAPhe became available, a number of investigations had probed the three-dimensional struct,ure of tRNA in solution. It turned out that these studies provided a considerable body of evidence that the structure of yeast tRNAPhe in solution is the same as that in the crystal (Rich & RajBhandary, 1976) with the possible exception of the anticodon loop. In the X-ray structure model the anticodon loop is highly structured. The five bases at the 3’ side of the loop are stacked upon each other. In order to accomodate this stack a sharp turn of the phosphate backbone is required. This is provided by the so called U turn. After Us3 (in the anticodon loop in yeast tRNAPhe, see Fig. 1) the phosphate P,, changes direction quite abruptly through a rotation of the P-O,, bond. The stabilization for this turn derives in part from a hydrogen bond formed between N,H of U,, and the phosphate P,,. It is interesting to note that U,,, which is located at the 5’ side of the anticodon, is an invariant base, i.e. a uridine is found in all other tRNAs at this position. This suggests that the anticodon conformation in the X-ray model of tRNAPhe may be a common feature of all other tRNAs. In this conformation U, 3 is not available for hydrogen-bonding to complementary oligonucleotides. Yet a number of oligonucleotide binding studies suggest that in solution it is available (Uhlenbeck et al., 1970; Eisinger & Spahr, 1973; Pongs et al., 1973;

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Pm. 1. (a) Cloverleaf structure of E. coli tRNA Phe (Barrel1 t Sanger, 1969). The structure of the hypermodified base ms2igA in the anticodon loop is given in Fig. 3; X stands for 3-N-(3.amino-3. carboxypropyl) uridine. (b) Cloverleaf structure of yeast tRNA Phe (RajBhandary & Chang, 1968). The structure of the Y-base in the anticodon loop is presented in Fig. 3.

Freier Q Tinoco, 1975; Yoon et al., 1975). Moreover, taking the X-ray structure as a starting point the results suggest that complementary oligonucleotides are able to induce a conformational transition of the anticodon loop. In theoretical models of protein synthesis (Woese, 1970; Lake, 1977) and theories of the development of protein synthesis during evolution (Crick et al., 1976; Eigen I.%Schuster, 1978) these conformational transitions play a crucial role. In the present paper we attempt to demonstrate by n.m.r.t methods that such transitions can indeed occur and investigate how these effects are transferred into the tRNA structure. In an earlier paper (Geerdes et al., 1978), we have shown that) resonances of protons hydrogen-bonded between the oligonucleotide U-U-C-A and the anticodon loop of yeast tRNAPhe are observable in the low-field ‘H n.m.r. spectra recorded at low temperature (2°C). These resonances may serve as internal probes of the anticodon loop structure in the presence of various complementary oligonucleotides. We were, however, not able to show whether the adenine residue in U-U-C-A was able to form a base-pair with U,, in the anticodon loop of yeast tRNAPhe. In this paper these investigations are extended by studying the binding of the oligonucleotides U-U-C-A, U-U-C-G and U-U-C-A-G to yeast tRNAPhe and of U-U-C-A to Escherichia c& tRNAPhe. The results indicate that the pentanucleotide U-U-C-A-G can form base-pairs with the base at the 5’ side of the anticodon in the anticodon loop. t See footnote

to p. 195.

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2. Materials and Methods Yeast tRNAPhe and E. coli tRNAPh” were purchased from Boehringer, Mannheim ; both tRNA species had an amino acid acceptance of about 1400 pmol/o.n.,,, unit, as determined by the manufacturer. samples were prepared by dialysing the tRNA according to Thiebe Mg 2 + -free tRNA (1975). Before use the tRNA was dialysed 4 times against a solution of 1 m&I-Na,S,O,. After dialysis the tRNA was lyophilized. The tRNA freed of Mg2+ contained 0.1 mol Mg2 +/mol tRNA as determined by atomic absorption spectrophotometry ; tRNA concentrations were calculated using 1 0.D.26,, unit/ml = 1.8 pM. The oligonucleotides U-U-C-A, U-U-C-G and U-U-C-A-G were synthesized according could be detected to the phosphotriester method (Van Boom et al., 1977a,b). No impurities by thin-layer chromatography, high-performance liquid chromatography and 31P n.m.r. A slight impurity (1%) was occasionally detected by lH n.m.r., giving rise to a resonance at 1.9 p.p.m. The oligonucleotides were used in the Na + form ; excess salt was removed by Sephadex GlO gel filtration. U-U-C-A, U-U-C-G and U-U-C-A-G concentrations were unit/ml as equivalent to 25.6 PM, 25.6 pM and 20.0 pM, respectcalculated using 1 o.D.~~~ ively. Appropriate quantities of oligonucleotide were lyophilized before addition to the tRNA samples. The n.m.r. samples were prepared by dissolving the lyophilized tRNA in O-2 ml H,O or 2H,0 buffer; solution conditions are indicated in the Figure legends. The H,O samples contained 5 o/0 2H20 to lock the field of the n.m.r. spectrometer to the resonance frequency of deuterium. lH n.m.r. spectra were recorded on a Bruker 360 MHz n.m.r. spectrometer, operating in the correlation spectroscopy mode; 500 to 2000 scans of 2 s each were accumulated in a Nicolet BNC 12 computer. The difference spectra were obtained by subtracting the tRNA spectrum recorded in the absence of oligonucleotide from the spectrum recorded in the presence of oligonucleotide. Occasionally, it was necessary to scale one of the spectra to be subtracted; this was done using the resonances at the low-field side and those at the high-field side as internal markers. Chemical shifts are indicated in p.p.m. down-field from the methyl resonance of the internal reference 4,4-dimethyl-4-silapentane-1-sulfonate. 40.5 MHz 31P n.m.r. spectra were recorded on a Varian XL-100 n.m.r. spectrometer operating in the FT-mode. Proton noise decoupling was used to remove the proton phosphorous J-couplings from the 31P spectrum ; the pulse angle used was 45°C. The field frequency ratio was stabilized by an internal deuterium lock. Chemical shifts are given with upfield shifts defined as negative. The with respect to the standard 80% H,PO, digital resolution was at least 1 point/Hz. Temperatures were held constant within 1-O deg. C for both n.m.r. spectrometers. The pH values of the solutions are not corrected for isotope effects and refer to the pH meter readings. Fluorescence binding experiments were carried out on a Perkin Elmer MPF 4 fluorescence spectrophotometer using the Y-base of yeast tRNAPhe as a probe. The excitation and emission wavelengths were set at 313 nm and 443 nm, respectively. Concentration of tRNA was 5 to 10 PM in a buffer containing 30 m&r-sodium phosphate, 100 mM-NaCl and 10 mM-MgCl, at pH 7.0 in 2Hz0.

3. Results (a) E#ect of oligonudeotide

binding on the methyl proton spectra of yeast tRNAPhe and E. coli tRNAPhe

In order to assess the mode of binding of the different oligonucleotides effect of binding on the tRNA structure, a number of experiments were

and the carried

out

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FIG. 2. 360 MHz proton n.m.r. spectra of the methyl protons in yeast tRNAPhe before (a) and after (b) addition of 1.7 mM-U-U-C-A. The signal from the C,, methyl group of the Y-base is shifted as indicated. The spectra were recorded at 35°C. Under the conditions used for spectrum (b) 59% of the anticodon sites are occupied. Asterisks indicate impurities. Solution conditions : 1 rn%! tRNB, 0.1 M-NaCl, 5 miw-Na,S,&, 10 miw-sodium phosphate, 10 m&l-Mg2+, pH 7.0 in aH,O.

using the methyl resonances of the tRNAs as probes. A representative example is given in Figure 2, which shows the 360 MHz lH n.m.r. spectra of the methyl group protons of yeast tRNAPhe before and after addition of U-U-C-A, recorded at 35°C. The two spectra are virtually identical except for the methyl resonance located at 2-O p.p.m. in the absence of the tetranucleotide (Fig. 2). This resonance shifts upfield upon complexation of U-U-C-A to yeast tRNAPha. A detailed assignment of the resonances given by Davanloo et al. (1979) is presented in Figure 1 of the accompanying paper. The positions of the methyl groups in the tRNA are presented in the cloverleaf structure in Figure l(a). The resonance at 2.0 p.p.m. can be assigned unambiguously to the C,, methyl group of the Y-base (Fig. 3) (Kan et nl., 1975,1977), which is located next to the anticodon (Fig. l(a)). As will be discussed below, the shift, can be taken as evidence for binding of U-U-C-A to the anticodon loop. The change in resonance position of the Y-base C,, methyl group at various concentrations of U-U-C-A can be used to calculate the binding constant of U-U-C-A to yeast tRNAPhe (Geerdes et al., 1978). This yields an association constant of 1050 M-l at 35°C in good agreement with the value of 1320 M-~ derived from the data of Pongs & Reinwald (1973).

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FIG. 3. (a) Structure of the hypermodified base, ms2i6A, (2.thiomethyl-W-isopentenyladenosine) in the anticodon loop of E. co& tRNAr”e. (b) Structure of the hypermodified Y-base, in the anticodon loop of yeast tRNAr”e.

Similar effects to those observed in Figure U-U-C-G and U-U-C-A-G are added to yeast tetranucleotides U-C-C-C and C-G-A-A, that loop, does not lead to changes in the methyl

base,

2 are obtained when the oligonucleotides tRNAPhe solutions, while addition of the are not complementary to the anticodon spectra.

(b)

w.m FIG. 4. 360 MHz spectra of the methyl protons in E. co& tRNAPhe before (a) and after (b) addition of oligonucleotide. The resonances of the thiomethyl group and the isopentenylmethyl groups of the msaiaA base are numbered 1 and 2, respectively; signal 3 is from the methyl group of T,,. Spectra were recorded at 16’C. Speotrum (b) was recorded after adding 2.6 mM-H-U-C-A. Asterisks indicate impurities. Solution conditions : O-4 mM-tRNA, 0.1 M-Nacl, 10 mM-Mgcl,, 6.5 mM-Na,S,O,, 30 mM-sodium phosphate, pH 7.4 in aH,O.

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Binding of the tetranucleotide U-U-C-A to E. coli tRNAPhe leads to changes similar to those observed in the methyl proton spectra of yeast tRNAPhe. E. co& tRNAPhe contains a moderate number of modified bases (see cloverleaf structure, Fig. l(b)). Of these bases only three have additional methyl groups. First there are the Ts4 in the T-loop and the m7G,, in the extra arm. These bases are found also in other tRNAs. In addition the base at the 3’ side of the anticodon is a highly modified adenine residue namely 2-methyl-thio-W-isopentenyl adenosine (ms2i6A) (see Fig. 3). The methyl proton spectrum of E. coli tRNAPhe between 0 and 2.5 p.p.m. is presented in Figure 4. Four methyl groups resonate in this spectral region: at 0.9, 1.7 and 2.4 p.p.m. On the basis of the assignments made in yeast tRNAPhe the resonance at 0.9 p.p.m. is attributed to the methyl group of T,,. Furthermore we do not expect a resonance of the m7G,, residue in this spectral region (Daniel & Cohn, 1975) and therefore the resonances at 2.4 and 1.7 p.p.m. can be assigned to the hypermodified ms2i6A,,. The resonance at 2.4 p.p,m. is attributed to the thiomethyl group and that at 1.7 p.p.m. to the two isopentenyl methyl groups. This follows directly from the relative intensity of the resonances. Furthermore the resonance position of the thiomethyl group is in the expected spectral region; e.g. a similar methyl group in methionine resonates at 2.2 p.p.m. (Varian NMR catalogue). Upon binding of U-U-C-A to E. co& tRNA Phe two resonances are affected, i.e. the resonance of the thiomethyl group is shifted upfield by 0.12 p.p.m., while one of the isopentenyl methyl resonances is shifted downfield by 0.06 p.p.m. Analogously to the findings with yeast tRNAPhe also in this case the methyl resonance of t,hymine 54 is not, affected. (b) Effect of oligonucleotide

binding

on the 31P spectrum of yeast tRNAPhe

31P n.m.r. spectra of tRNAs contain a number of well-resolved resonances shifted outside the main resonance at 0 p.p.m. (Gueron & Shulman, 1975). The resonance position of an internucleotide phosphate is thought to depend on the geometrical arrangement of the atoms around the phosphate, i.e. the chemical shift of a phosphate resonance depends on the 0,.--P-O,. dihedral angle, on the torsional angles around the 0,.-P and P-O,, bonds and possibly also on the presence or absence of hydrogen bonds with the internucleotide phosphates (Gorenstein et al., 1976 ; Salemink et al., 1979). Figure 5 shows the effect of interaction of yeast tRNAPhe with U-U-C-A on the 31P n.m.r. spectrum. Upon complex formation of yeast tRNAPhe with U-U-C-A two resonances shift downfield, which have been numbered 1 and 2. The shift of resonance 1 is small (0.2 p.p.m.), but resonance 2 shifts to lower field by an amount of 0.55 p.p.m. after full saturation of the anticodon sites. In a more extended titration experiment the shift of this resonance was used to derive an association constant for the equiliat 35°C. This is in good brium between tRNA and U-U-C-A, yielding 1200 M-~ agreement with the value obtained at this temperature from the shifts of the Y-base C,, methyl resonance and also with values derived by other methods (wide sup”). An unambiguous assignment of the internucleotide phosphate resonances of yeast tRNAPhe is not yet available, but it is worthwhile mentioning that the same resonances are affected by modifications of the anticodon loop. After enzymatic nicking of this loop these resonances merge with the main resonance at about 0 p.p.m. (Salemink

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p.p.m. FIG. 5. The effect of addition of U-U-C-A on the 40.5 MHz 31P n.m.r. spectrum of yeast tRNAPhe. Spectra were recorded at 35°C in the absence (a) and in the presence of U-U-C-A (1.1 rn~ in (b) and 3.6 rn~ in (c)). Spectra (b) and (c) represent situations in which 39% and 79% of the antioodon sites are occupied, respectively. The vertical scale of the spectra on the left side is 6 times larger than that on the right side. Asterisks indicate the resonances coming from the 3 internucleotide phosphates of U-U-C-A. Resonances numbered 1 and 2 are from the tRNA and are shifted as a result of oligonucleotide binding. Solution conditions: 0.9 mna-tRNA, 10 mM-MgCl,, 100 mMN&l, 1 mM-EDTA, 12 mm-Na,S,O,, 30 miw-sodium cacodylate at pH 7.2 in 2Ha0.

et al., 1979). In principle it is possible that these resonances come from phosphates far removed from the codon binding sites because long-range effects may influence their resonance positions. However, this explanation is rendered unlikely on the basis of 31P n.m.r. experiments of yeast tRNA Phebefore and after modification by RNase T, (Salemink et al., 1979). Therefore it is reasonable to assign resonances 1 and 2 to internucleotide phosphates within the anticodon loop of the tRNA. This means that

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p.p.m. BIG. 6. 360 KHz n.tn.~. spectra of the hydrogen-bonded protons in yeast tRNAPbe before (ct) and after (b) addition of U-U-C-A up to a concentration of 6.1 mu. Spectra were recorded at, 35%‘. Under the conditions used for spectrum (b), 86% of the anticodon sites are occupied. tRNA concentration was 1.26 mM in H,O. Other solution condkions are indicated in the legend to Pig. 2, (b) - (a] represents the difference spectrum except that no Mgz+ was present. The spa&urn obtained by subtracting (a) from (h).

a change in the conformation U-U-C-A.

of the anticodon loop is induoed upon binding of

(c) E#ect of U-U-G-A binding on the hydrogen-bonded proton spectra of yeast tRNAPhe and E. coli tRNAPh” The effect of addition of U-U-C-A on the hydrogen-bonded proton spectrum of yeast tRNAPh” is shown in Figures 6 and 7. Figure 6(a) and (b) presents the tRNA spectra, recorded at 35°C in the absence and presence of U-U-C-A, respectively. In the latter situation 86% of the anticodon sites are occupied. The difference between these two spectra is given at the bottom of Figure 6. The most important change between the two spectra is found around 12.6 p.p.m. The most likely explanation of this difference is that, upon complex formation, a resonance shifts from 12G to 12-6 p.p.m. In addition a slight change is observable at 10.6 p.p.m.

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ppm FIG. 7. 360 MHz n.m.r. spectra of the hydrogen-bonded protons in yeast tRNAPhe, before (a) at and after (b) addition of U-U-C-A up to a concentration of 1.8 mM. The spectra were recorded 2°C. Under the conditions used for spectrum (b), 97% of the anticodon sites are occupied. tRNA concentration was 1.26 rn~ in H,O. Other solution conditions are given in the legend to Fig. 2, except that no Mg2+ was present. The spectrum (b) - (a) represents the difference obtained by subtracting (a) from (b).

At 2°C at an occupancy of 970/b of the anticodon sites, the G(N1) and U(N3) protons hydrogen-bonded between the oligonucleotide and the tRNA become visible. This is shown in Figure 7, where the spectra in the absence (Fig. 7(a)) and in the presence (Fig. 7(b)) of U-U-C-A are presented. Again a change is observed around 125 p.p.m. analogously to that at 35°C. In addition, extra resonance intensity is found at 13.3 p.p.m. and at 113 p.p.m. The signal at 13.3 p.p,m., which corresponds to at least two protons, has been assigned to the hydrogen-bonded U(N3) protons of A*U pairs and the signal at 1143 p.p.m. is thought to be generated by the G-C pair formed after binding of the tetranucleotide to the tRNA (Geerdes et al., 1978). At

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P.P.m FIG. 8. 360 MHz n.m.r. spectra of the hydrogen-bonded protons in E. coli tRNAPhB before (a) and after (b) the addition of 4.7 mu-U-U-C-A, recorded at 35°C. Under the conditions used for spectrum (b), 84% of the anticodon sites are occupied. Solution conditions: 0.9 m&X-tRNA, 0.1 iv-Nacl, 10 mM-MgCl,, 13 miw-Na&O,, 30 mna-sodium phosphate at pH 6.8. The difference spectrum at the bottom of this Figure shows spectrum (b) minus spectrum (a).

11.4 p.p.m. we find a reproducible increase in intensity, which we cannot explain. The change observed around 10.6 p.p.m. at 35°C is more clearly visible at 2°C. Also the low-field n.m.r. spectra of the hydrogen-bonded protons of E. coli tRNAPhe were recorded at various temperatures in the absence and presence of U-U-C-A. An example of the effect of binding of U-U-C-A on the spectrum recorded at 35°C is shown in Figure 8. Figure 8(a) and (b) presents the spectra in the absence and in the presence of U-U-C-A, respectively. Under the conditions of Figure 8(b) 84yG of the tRNA is estimated to be complexed with U-U-C-A. The difference spectrum at the bottom of this Figure shows that after binding slight shifts of resonances can be observed, of which we consider the one around 13.2 p.p.m. as the most important. We interpret this shift as a slight change of the position of a resonance, originally at 13.2 p.p.m. and after binding at 13.0 p.p.m. This is more clearly visible in the spectra recorded at 1°C (see Fig. 9). It is also evident, from the difference spectrum in Figure 8, that some resonance intensity appears at 10.5 p.p.m. upon oligonucleotide binding. These findings are similar to those for yeast tRNAPhe except that we now see a shift at 13.2 p.p.m. instead of 12.5 p.p.m. As will be discussed below, we attribute this effect to the presence and absence of Mg2 + in the E. coli tRNAPhe and yeast tRNAPhe samples, respectively (see legends to Figures). At l”C, at an occupancy of 98% of the anticodon sites, the resonances of the G(N1) and U(N3) protons hydrogen-bonded between the tRNA and U-U-C-A are visible in the spectrum (Fig. 9) as in the yeast tRNAPhe-U-U-C-A complex. As shown in the difference spectrum at the bottom of Figure 9, extra resonance intensity corresponding to two or three proton resonances appears at 13.7 p.p.m. and intensity

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p.p.m.

9. 360 MHz n.nu. spectra of the hydrogen-bonded protons in E. coli tRNAPhe before (a) and after (b) addition of 1.3 mu-U-U-C-A, recorded at 1’C. Under the conditions used for spectrum (b), 98% of the anticodon sites are occupied. For other details see legend to Fig. 8. FIQ.

corresponding with one proton resonance appears at 11.8 p.p.m. In analogy with the assignments made for the yeast tRNA Phe-U-U-C-A complex these resonances are attributed to the hydrogen-bonded ring nitrogen protons of two or three A *U pairs and one G*C pair, respectively. In addition to the new resonances others are shifted. We have discussed already the shift of the resonance at 13.2 p.p.m. In addition & shift is observed at 11.1 p.p.m. It is also noted that an increase of resonance intensity at 105 p.p.m. at 35°C is more outstanding at 1°C. It is at these positions that we also see changes in the spectra of yeast tRNAPhe. (d) Biding

of U-U-C-G

and U-U-C-A-G

to yeast tRNAPhe

From the experiments with U-U-C-A we cannot be sufficiently sure that a base-pair is formed between U,, in the tRNA and the adenine residue in the tetrsnucleotide, although the difference spectrum in Figure 9 obtained for the E. coli tRNAPheU-U-C-A complex strongly suggests such a conclusion. This possibility was checked by binding U-U-C-G and U-U-C-A-G. For U-U-C-G we would then expect the formation of a G-U base-pair between U,, and the G in the tetranucleotide. The resonances of such a base-pair are observable (Baan et al., 1977; Johnston & Redfield, 1978) and their positions can be estimated (Geerdes & Hilbers, 1979). In a series of experiments, similar to those described above for U-U-C-A, difference spectra were obtained for the U-U-C-G complex at 35°C and 1°C but no resonances from a G-U base-pair

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p.p.m. FIG. 10. 360 MHz n.m.r. spectra of the hydrogen-bonded protons in yeast tRNAPhe before (a) and after (b) the addition of 3.8 mu-U-U-C-A-G, recorded at 1°C. Under the conditions used for spectrum (b), 99% of the anticodon sites are occupied. The spectrum at the bottom of this Figure represents spectrum (b) minus spectrum (a). Solution conditions: 1 miw.tRNA, 0.1 iv-NaCl, 24 mM-Na&O,, 30 m&r-sodium phosphate, pH 7.

could be detected, the other features being similar to the difference spectra-obtained for U-U-C-A. Subsequently we looked at the binding of U-U-C-A-G to yeast tRNAPhe. This pentanucleotide is complementary to the five bases of the 5’ side of the anticodon loop. At 35°C again a slight shift is induced in the position of a resonance at 125 p.p.m, Some extra resonance intensity also appears at 10.7 p.p.m. Both features were observed in the U-U-C-A experiment. At low temperatures (i.e. at 1%) resonances of the G(N1) and U(N3) protons hydrogen-bonded between the tRNA and the pentanucleotide become manifest in the spectrum. This is shown in Figure 10, where the spectra of the tRNA in the absence (Fig. 10(a)) and presence (Fig. 10(b)) of 3.8 mM-U-U-C-A-G are presented.

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FIG. 11. 360 MHz n.m.r. spectra of the hydrogen-bonded protons in yeast tRNAPhe before (a) and after (b) the addition of 3.8 mM-U-U-C-A-G, recorded at 15’C. Under the conditions used for spectrum (b), 99% of the anticodon sites are occupied. The spectrum at the bottom of this Figure represents spectrum (b) minus spectrum (a). For further details see legend to Fig. 10.

As with the other nucleotides the difference spectrum at the bottom of Figure 10 shows extra resonances appearing after complex formation accompanied by shifts of some other resonances. The changes around 12-5 p.p.m. and 10.7 p.p.m. observed at 35°C are retained at 1°C. In order to obtain a quantitative estimate of the increase of hydrogen-bonded protons, the difference spectrum (Fig. 10) was integrated. This requires that a baseline be drawn in the difference spectrum to which the following criteria were applied: (1) the high and low-field parts of the difference spectrum are described by a stochastic process; the average value of these signals must be zero.

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(2) In all our difference spectra a difference of two Lorentzian lines, slightly shifted with respect to each other, is observed around 12.5 p.p.m.; integration of both parts of this signal should yield zero intensity. Using these criteria the baseline has been drawn. This yields an intensity increase of four protons between 13.0 and 13% p.p.m. I n accordance with the assignments made with an estimated accuracy of f300/. above three of these resonances are attributed to A*U pairs, i.e. A*U,,, A*U,, and while the fourth resonance may come from G.C,, as will be discussed below. A.U,,, Around 11.6 p.p.m. increase of resonance intensity is observed. The signal, however, is rather broad. In accordance with the earlier assignments it is attributed to the G. C,, base-pair, although we have no explanation as to why it is broader in t,his particular experiment. Experiments were carried out also at 15°C. At this temperature resonance intensity from hydrogen-bonded protons of the tRNA-pentanucleotide complex is still manifest in the spectrum, i.e. at 13.3 and 11.4 p.p.m. (Fig. ll), though the total intensity is reduced as compared with the experiment performed at 1°C. Interestingly, the resonance at 11.9 p.p.m. assigned to G*C,, now has its normal intensity. (e) Fluorescence binding

studies

The n.m.r. studies of the tRNA-U-U-C-A-G complex were supplemented by fluorescence binding studies. The quantum yield of the fluorescence of the Y-base of yeast tRNAPhe decreases upon binding of oligonucleotides to the anticodon (Eisinger et al., 1970). Changes in the fluorescence intensity were determined as a function of the ratio, U-U-C-A-G/tRNA, to derive the binding constant at different temperatures. and At 4°C and at 25°C the association constants amounted to 2.5~ lo5 M-~ 1.0 x lo4 M-l, respectively. The value for the association constant at 4°C is in reasonable agreement with the results of Eisinger & Spahr (1973), who estimated it to be equal to or higher than 3 x lo5 M-~ at this temperature. From the temperature dependence of the binding constant the reaction enthalpy and entropy were determined, yielding -25 kcal.mol-l and -66 cal.mol-l.deg.-l, respectively.

4. Discussion (a) Do the oligonudeotides bind to the anticodon loop? The experiments described in this paper were undertaken to investigate the effect of codon-anticodon interaction on the structure of tRNAPhe. It is crucial to such a study that one can be sure that the oligonucleotides used are really binding to the anticodon. The experiments presented here provide strong evidence that this is indeed the case. Upon binding of U-U-C-A, U-U-C-G and U-U-C-A-G to yeast tRNAPhe only the resonance of the C,, methyl group of the Y-base exhibits an observable shift (Fig. 2). Although one could argue that binding might take place at a different site, thereby shifting the C,, methyl resonance as a result of a longdistance effect, this is rendered highly unlikely because the resonances of the other methyl groups do not show any shifts at all, while except for the acceptor stem the methyl groups are distributed more or less evenly over the molecule. Moreover. addition to a tRNA sample of the nucleotides C-G-A-A or U-C-C-C, which are not

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complementary to the anticodon, did not yield any changes in the position of the C,, methyl resonance. Similar behaviour is observed when U-U-C-A is complexed to E. c&i tRNAPhe (Fig. 4). Although this tRNA differs in 29 of the 76 nucleotides in the primary sequence (Fig. l), only the resonances of the hypermodified base next to the anticodon are affected by binding of U-U-C-A (Fig. 4) and not the thymine methyl resonance. Additional evidence that the oligonucleotides bind to the anticodon is provided by the 31P n.m.r. experiments. U-U-C-A binding induces shifts of resonances in the of the 31P n.m.r. spectrum of yeast tRNAPhe, which are also affected by modifications anticodon loop of the tRNA. Moreover, the binding constants obtained from these shifts as well as from the C,, methyl resonances of the Y-base were found to be equal within experimental error, indicating that the same binding process is apparent from the ‘H n.m.r. as well as the 31P n.m.r. experiments. These arguments taken together are considered sufficient evidence that binding takes place at the anticodon. (b) InJluence

of magnesium

ions

A number of experiments on yeast tRNA Phe, discussed in Results, were performed on samples that were freed from Mg 2+. We want to emphasize that for U-U-C-A these experiments were also performed in samples containing 10 m&r-Mg2+ (total concentration). The results obtained in both sets of experiments were essentially equivalent. The spectra recorded in the presence of Mg2+ were as a rule somewhat less well-resolved than those obtained in the absence of Mg2 + . This is particularly true for the hydrogen-bonded proton spectra recorded at low temperatures. This can be seen from a comparison of the spectra of yeast tRNAPhe in Figure 7 recorded in the absence of Mg2+ at 2”C, and the spectra of E. coli tRNAPhe in Figure 9 recorded in the presence of Mg2 + at 1“C. The conclusions we arrived at for E. coli tRNAPhe are only applicable to samples containing Mg2 + . It has been shown that E. co.5 tRNAPhe can adopt different strucform is found in the absence tures depending on solution conditions. A “denatured” of Mgl+” which has a structure different from the native form present in Mg’+containing solutions (Streek & Zachau, 1972). These differences become manifest in the hydrogen-bonded proton spectra (not shown). (c) Structure

of the oligonucleotide-anticodon

loop complexes

In an early attempt to obtain information on the structure of the anticodon loop in solution, Eisinger $ Spahr (1973) studied the complex formation of a digest of in 23 S ribosomal RNA from E. coli with yeast tRNA Phe. One of the oligonucleotides this digest is the pentanucleotide U-U-C-A-G. The authors demonstrated that this oligonucleotide binds to yeast tRNAPhe with an association constant equal to or higher than 3 x lo5 M-l at 4”C, and they concluded that five base-pairs are formed in the tRNA pentanucleot,ide complex and the anticodon loop is in the 5’ stacked conformation. The experiments described in this paper support their conclusions in much more detail. Four to five hydrogen-bonded resonances from ring nitrogen protons are observed in the tRNA-U-U-C-A-G complex, so that four to five basepairs are formed between the anticodon loop and the pentanucleotide.

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1 (a)

(b)

FIG. 12. Schematic representation of the anticodon loop conformations of yeast tRNAP”“. The anticodon sequence GAA is drawn in the white box. The structure in (a) is the 3’ stacked conformation, which is representative of the X-ray structure model of yeast tRNAP”*. In (b) the pentanucleotide U-U-C-A-G is bound to the anticodon loop in the 5’ stacked conformation. (-----) Connection between bases in loop section.

This conclusion is supported by the fluorescence-binding experiments. The thermodynamic parameters for binding of U-U-C-A to yeast tRNAPhe have been determined by Yoon et al. (1975); they found -19&4 kcal.molll and -45&6 calmol-l.deg.-l for the enthalpy and entropy of complex formation, respectively. Formation of an extra G-C pair in the U-U-C-A-G-tRNA complex compared with the U-U-C-AtRNA complex is expected to change the enthalpy and entropy terms by -6 kcalmol - l respectively (Borer et al., 1974). Combining these values and -13 cal.mol-l.deg.-l, we expect to find, for the enthalpy and entropy of formation of the U-U-C-A-G-25&4 kcal.mol-l and --58-&5 cal.mol-l.deg.-l, respectively. tRNA complex, These values are in good agreement with those experimentally observed, i.e. -25 kcal.mol-l for the reaction enthalpy and -66 cal.molll.deg.-l for the reaction entropy. In order that a five-base-paired complex can be formed the anticodon loop most likely adopts a conformation in which the five bases at the 5’ side are stacked upon each other, i.e. the anticodon loop adopts a so-called 5’ stacked conformation contrary to the 3’ stacked conformation of the crystal structure model (Fig. 12). Assuming that in the 5’ stacked conformation the tRNA-U-U-C-A-G complex forms an A’ RNA double helix (Arnott, 1971) the resonance positions of the hydrogen-bonded protons can be predicted by calculating the ring-current shifts, The results are listed in Table 1 together with the observed resonance positions. In addition calculated resonance positions are given for those hydrogen-bonded protons that can be formed in the 3’ stacked conformation. To this end the co-ordinates of

212

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ET

AL.

1

Comparison of the observed resonance positions (p.p.m.) of the hydrogen-bonded G(N1) and U(N3) protons in the U-U-C-A-G-yeast tRNAPhe complex with the positions predicted on the basis of the two alternative structures of the anticodon loop (5’ stacked and 3’ stacked, Fig. 11) Base-pair between oligonucleotide and tRNA

Positions Observed

u. A,,

13.3 13.3 11.6 13.6 13.1 (?)

U.&s C.‘&, A.U,, G.C,, Ring-current shifts of the 5’ Shulman et al. (1973). Ring-current Hilbers (1977) using the crystal p.p.m. for an A.U pair and 13.6 t Ring-current shift of the A,,

Predirt,ed 5’ stacked 144 13.3 11.6 13.5 13.5t

3’ stacked 14.4 14.4 11.6-12.6 Impossible Impossible

stacked conformation were calculated using corrected table of shifts of the 3’ conformation were calculated as by Geerdes & co-ordinates of Jack et nl. (1976). Intrinsic positions were 14.5 p.p.m. for a G.C pair. . Y39 base-pair is not taken into account.

the crystal structure (Jack et al., 1976) were used together with ring-current shift 1977). Comparison of the resonance positions calculations (Geerdes & Hilbers, calculated for the 5’ stacked conformation with those observed shows that the two sets of data are in reasonable agreement except for the A*U,, base-pair. However, this base-pair is at the end of a double helix (Fig. 12) and an upfield shift may be found as a result of fraying effects, which, as demonstrated earlier, may occur at the end of short double helices (Pate1 & Hilbers, 1975). In the 3’ stacked conformation the base-pairs A*U,, and GC,, cannot be formed. Moreover, the observed position considerably deviates from the predicted positions. Therefore it may be of A-U,, that the structure of the anticodon loop in the tRNA-U-U-C-A-G complex is similar to an A’ RNA or A RNA helical structure with the five bases on the 5’ side of the anticodon loop forming a continuous stack.

TABLE

2

Resonance positions (p.p.m.) of hydrogen-bonded protons in the co&n-antic&n complex observed in the various tRNA&gonucleotide conbplexes tRNA-oligonucleotide

Yeast Yeast E. coli Yeast

G.C,,

Observed positions A.U3, and A.Us6

tRNAPhB-U-U-C-A tRNAPhB-U-U-C-G tRNAP”“-U-U-C-A

11.8 12.1 11.8

13.3 13.3 13.7

tRNAPhB-U-U-C-A-G

11.6

13.3

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Subsequently, it is interesting to compare the results obtained for the tetranucleotides U-U-C-A and U-U-C-G with those of the pentanucleotide. To this end the resonance positions of the hydrogen-bonded protons in the various oligonucleotidetRNA complexes are listed in Table 2. In all complexes resonances are observed around 13.4 p.p.m. and 11.8 p.p,m. In addition the changes in the 31P spectra of yeast tRNAPhe after complex formation are highly similar for all oligonucleotides studied, while for the three oligonucleotides whose binding has been followed via the methyl proton spectrum the shift of the C1, methyl of the Y-base is similar. This indicates that in these complexes the anticodon loop conformation is similar to that found in the pentanucleotide-tRNA complex, i.e. U,, most likely binds to A or G in the U-U-C-A and U-U-C-G complexes, and not to P,,. Accordingly the increased binding constants of the tetranucleotides U-U-C-A and U-U-C-G (Pongs & Reinwald, 1973) relative to the binding constant of UUC are most naturally explained by the formation of a fourth base-pair a,s originally proposed (Uhlenbeck et al., 1970). In the X-ray diffraction model the anticodon loop is in a 3’ stacked conformation and there has been tendency to explain the increased binding of the tetranucleotides to this conformation as due to the stabilizing contribution of the dangling ends. In view of the results presented above this seems an unlikely explanation for the complexes studied here. (d) Changes in tRNA structure Although we were not able to detect any gross conformational changes in the tRNA molecule after binding of the oligonucleotides, the n.m.r. experiments do indicate that smaller changes take place. The shifts observed in the 31P spectra after complex formation show that changes in the anticodon loop occur. This is corroborated by the observation that the shifts of the methyl resonances in yeast tRNAPhe and E. wli tRNAPhe are too large to be caused by ring currents from the U residue in the A.U,, base-pair. They may be caused by an improved stacking of one of the neighbouring adenine residues. Subsequently it is interesting to see whether t.he changes in anticodon loop conformation are propagated into the tRNA molecule. Indeed, in the low-field n.m.r. spectra of yeast and E. wli tRNAPhe resonances from the tRNA are influenced by oligonucleotide binding. A shift of a resonance located at 13.2 p.p.m. is observed upon binding of U-U-C-A to E. coli tRNAPhe (Figs 8 and 9). This resonance may come from the A. Y base-pair, which is adjacent to the anticodon loop. In similar experiments using yeast tRNAPhe a resonance located at 12.5 p.p.m. is affected by oligonucleotide binding; we speculate that this resonance comes from the G,,.C,, base-pair, which is adjacent to the A. Y pair. The shift of these resonances indicates that slight changes in the tRNA structure indeed occur. Additional indications for alterations in the tRNA structure come from the spectral region between 10 and 11.5 p.p.m. Slight shifts and additional resonances are observable after complex formation of the tRNAs with U-U-C-A and U-U-C-A-G. These spectral changes were reproducible and although unfortunately we cannot assign these effects to particular structural changes in the molecule, they provide a qualitative confirmation of the conclusions above. It has been suggested earlier (Schwarz et al., 1976; Schwarz & Gassen, 1977) that a disruption of the tertiary hydrogen bonds between the D-loop and the T-loop is

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triggered by codon-anticodon interaction. The present results provide no evidence that such an event does occur after complex formation with U-U-C-A and U-U-C-A-G. Since the conclusions of Gassen’s group were based on the observation that C-G-A-A which is complementary to the T-F-C-G sequence, binds to the tRNA-codon complex (tRNAPhe-(pU)8) but not to tRNAPhe alone, we performed the n.m.r. equivalent of these experiments. It followed that we could not detect any binding of C-G-A-A to the tRNA either in the absence or in the presence of oligonucleotides complementary to the anticodon (Geerdes, 1979).

(e) Comparison

with results from other methods

A number of oligonucleotide binding studies have indicated that the anticodon loop can be forced rather easily into conformations strongly deviating from the 3’ stacked conformation. Apart from the U-U-C-A-G binding to yeast tRNAPhe found by Eisinger & Spahr (1973), it was observed that trinucleotides, being complementary to one or two bases at the 5’ side of the anticodon and two or one bases of the anticodon, respectively, bind to E. coli tRNA fMet (Freier & Tinoco, 1975; Pongs et al., 1973), while such a binding is not possible in the X-ray diffraction model of yeast tRNAPhe. Moreover, Yoon et al. (1975) in their study of the binding of UUC and U-U-C-A to yeast tRNAPhe found a decrease in the rotational strength of the fluorescence-detected dichroism of the Y-base. From this, they concluded that the Y-base cannot be rigidly stacked on the oligonucleotides when bound to the tRNA, as is expected in a 3’ stacked configuration. Rather, in the complex the Y-base seems to be much more flexible than in the 3’ stacked conformation, In this respect it is interesting to consider recent experiments by Davanloo et al. (1979), who studied the formation of the yeast tRNAPhe-E. coli tRNAG1” complex employing the IH n.m.r. spectrum of the methyl groups in these tRNAs. The upfield shift of the C,, methyl proton resonance amounts to 0.25 p.p.m., which is about twice the value observed in our experiments, suggesting an improved stacking of the Y-base relative to the adjacent adenines in the yeast tRNAPhe-E. coli tRNAGIU complexes. Apparently, the structure of the anticodon loop in this complex differs from that in the oligonucleotide-yeast tRNAPhe complex. It is clear from the above discussion that the structure of the anticodon loop in solution cannot be identified with one particular conformation and depends on solution conditions. There are some other observations supporting this notion. Urbanke & Maass (1978) detected a conformational transition in the anticodon loop by fluorescence-detected temperature-jump techniques. Moreover, it was noted that in the crystal structure of yeast tRNA Phe the anticodon nucleotides have a relatively differences between the large thermal motion (Holbrook et al., 1978). Also important anticodon loop conformation of yeast tRNAPhe and E. coli tRNAfMet were detected by X-ray diffraction studies (A. Rich, personal communication). Depending on solution conditions and complex formation, if any, the anticodon loop can be forced into a particular conformation and therefore seems rather flexible. This is clearly demonstrated in the n.m.r. spectra, which show that binding of the oligonucleotides changes the anticodon loop conformation as is manifest from the shifts of the Y-base C1 1 methyl resonance and internucleotide phosphate resonances.

CODON-ANTICODON

(f) Xignifcunce

INTERACTION.

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215

with respect to protein synthesis

The present n.m.r. study has demonstrated that resonances of protons hydrogenbonded between the anticodon loops and the complementary oligonucleotides can be detected. Since this method provides enough resolution to be able to distinguish resonances of individual protons in the tRNA-oligonucleotide complex, we are now in a position to discuss how the binding of the complementary oligonucleotides to the anticodon loop may mimic the codon-anticodon recognition process on the ribosome. In the present models of protein synthesis the ribosoma,l A and P-sites are occupied simultaneously by a tRNA molecule (Liihrmann et aE., 1979). The tRNA in the P site has attached to it the growing polypeptide chain, while the tRNA molecule in the A site is charged with the amino acid, which is connected to the peptide chain in the process of transpeptidation. Hence during one step in these series of events the CCA ends of both molecules must be close enough together in order to allow the transpeptidation of the amino acid attache’d to the tRNA in the A site. At the same time, the bases of the anticodons of the two tRNAs must form six consecutive base-pairs in order t,o maintain the proper reading frame. These requirements impose severe constraints as to the spatial positioning of the two tRNAs and the mRNA, and several models have been proposed in which it is assumed that during the elongation process a transfer from the 3’ to the 5’ stacked conformation takes place (Woese, 1970: Crick et aE., 1976; Lake, 1977). Our own results as well as other oligonucleotide binding experiments indicate that t’he anticodon loop can adopt the 5’ stacked conformation and that it is flexible. The observation that the pentanucleotide U-U-C-A-G can form base-pairs with the bases at the 5’ side of the anticodon

demonstrates

that

the invariable

residue

U,,

is avail-

able for hydrogen-bonding with complementary oligonucleotides. Subsequently, it is interesting to ask whether this residue is also available for basepairing to the mRNA on the ribosome. There are indications that such base-pair formation can in fact take place (Taniguchi & Weissman, 1978 ; Manderschied et al., 1978: Riddle & Carbon, 1973). However, critical examination of these results shows that only a small percentage of tRNAs will form a fourth base-pair with the messenger (Kurland, 1979; Geerdes, 1979). Hence although oligonucleotide binding experiments and the n.m.r. experiments indicate that base-pairs can easily be formed between bases on the 5’ side of the anticodon and a complementary oligonucleotide, this may not so easily happen on the ribosome. Rather the positioning of the codon triplet, seems to be critical or the anticodon loop is kept in a particular conformation, so that such interactions are avoided.

We are much indebted to Mrs G. Wille Hazeleger for the synthesis of the oligonucleotides; to E. Kremer, I. Vermolen-Van Bentem and J. van Kessel for experimental assistance. We thank Dr J. Walters for his help with the fluorescence binding experiments. We gratefully acknowledge Stichting voor Zniver Wetenschappelijk Onderzoek for support of the 360 MHz n.m.r. facility at Groningen and Dr R. Kaptein and K. Dijkstra for keeping the instrument in excellent condition.

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