Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA

J.

Mol. Biol. (1992) 227, 1173-1181

Correlations Between Fluorine-19 Nuclear Magnetic Resonance Chemical Shift and the Secondary and Tertiary Structure of 5FluorouraciLsubstituted tRNA Wen-Chy Chu, Agustin Kintanar and Jack Horowitz7 Department of Biochemistry and Biophysics and the Molecular, Cellular and Developmental Biology Program Iowa State University, Ames, IA 50011, U.S.A. (Received 5 March

1992; accepted 26 June 1992)

To complete assignment of the 19F nuclear magne t’K resonance (NMR) spectrum of 5-fluorouracil-substituted Escherichia coli tRNAVal, resonances from 5-fluorouracil residues involved in tertiary interactions have been identified. Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed. FU54 and FU55 were identified by 19F homonuclear Overhauser experiments and were then assigned by comparison of their 19F NMR spectra with those of 5-Auorouracil-labeled yeast tRNAPh” mutants having FU54 replaced by adenine and FU55 replaced by cytosine. FU8 and FU12, were assigned from the 19F NMR spectrum of the tRNAVal mutant in which the base triple GQ-C23-G12 substituted for the wild-type AQ-A23-FU12. Although replacement of the conserved U8 (FUS) with A or C disrupts the tertiary structure of tRNAVal, it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme. Analysis of the “F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAV”’ indicated a strong correlation to tRNA secondary and tertiary structure. 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5fluorouracil. However, the signal from FU59, in the T-loop of tRNA’“‘, was shifted more than 1 p.p.m. downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops. The 19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m. range, when fluorouracil was base-paired with adenine in helical stems. This upfield shift was less pronounced for the fluorine of the FU7. A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the An unanticipated finding was that the 19F continuous acceptor stem/T-stem helix. resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m. downfield of those from fluorouracil residues paired with A. In the 19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.

Keywords: lgF nuclear

magnetic tertiary

resonance; tRNA structure; structure; chemical shift

1. Introduction Our

studies with 5-fluorouracil (FUra$)-labeled coli tRNAV”’ and other fluorinated tRNAs have demonstrated that 19F NMR spectroscopy is a powerful tool for monitoring tRNA structure in solution (Hills et al., 1983; Gollnick et aE., 1986, 1987; Hardin et al., 1986, 1988; Hardin &

Escherichia

t Author addressed.

to whom

all correspondence

should

be

1173 002%2836/92/20117349

$08.00/O

Horowitz, 1991a,b). complete

secondary

structure;

1987; Chu & Horowitz, 1988, 1989, Interpretation of the results requires assignment of the spectrum, but this has

f Abbreviations

used: FUra, 5-fluorouracil;

(FUra)tRNA Va’, 5-fluorouracil-substituted E. coli tRNA”“‘; (FUra)tRNAPh”, 5-fluorouraeil-substituted yeast tRNAPh’; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; SIX, solvent isotope shift; VRS, valyl-tRNA synthetase; p.p.m., parts per million. 0 1992 Academic

Press Limited

W.-C. Chu et al.

1174

been difficult to accomplish. A variety of chemical and physical strategies have been used to make preliminary assignments of several resonances in the 19F NMR spectrum of fluorine-substituted E. coli tRNAva’ (Gollnick et al., 1986, 1987; Hardin et al., 1986, 1988). Recently, by systematically replacing individual FUra residues in the tRNA, we were able to assign resonances corresponding to FUra residues in loop and stem regions (Chu & Horowitz, 1989; Chu et al., 1992, accompanying paper). However, we encountered difficulties in applying this strategy directly to the assignment of FUra residues involved in tertiary interactions. These tertiary nucleotides, many conserved in all elongator tRNAs, play an important role in maintaining proper tRNA folding. Nucleotide substitutions that prevent or limit tertiary interactions disrupt tRNA structure, thereby dist’orting the 19F NMR spectrum and making assignments difficult. Certain substitutions can, however, be tolerated with little effect on tertiary folding and the aminoacylation activity of tRNA (Robertus et al., 1974; Klug et al., 1974; Sampson et al., 1990). By characterizing the 19F NMR spectra of such mutant tRNAs, by 19F homonuclear Overhauser techniques, appbing and by comparing the 19F NMR spectrum of E. co& with those of wild-type and mutant (FUra)tRNAv”’ yeast (FUrajtRNAPh”, we have succeeded in the peaks from tertiary fluorouracil assigning residues in (FUra)tRNAVa’, thus completing assignment of all 14 resonances in the “F NMR spectrum of the tRNA. The results reveal several interesting correlations between the structural environment of FUra residues in the tRNA molecule and the chemical shift position of the corresponding resonances. Although replacing the conserved uracil-8 (FUS) disrupts tbe tertiary folding of (FUra)tRNAVa’, the mutant tRNAs retain appreciable aminoacylation activity. Their affinity for valyl-tRNA synthetase (VRS) is decreased, i.e. the K, value for the tRNA increases, but there is only a small decrease in k,,,.

2. Experimental

Procedures

Wild-type and mutant forms of FUra-substituted tRNA”“” were prepared, as described (Chu & Horowitz, 1989), by bacteriophage T7 RNA polymerase-catalyzed in vitro transcription of recombinant phagemids containing the E. coli tRNA”“’ gene. Mutant tRNAs are designated by the letter and position number of the base in the wildtype sequence that has been altered, followed by the base that has been introduced, e.g. tRNAVa’-FUSG has the FUra at position 8 replaced by guanine. SFluorouracilsubstituted yeast tRNAPh”, as well as the FU54A and FU.55C variants, were transcribed by similar procedures from recombinant plasmids kindly supplied by Dr 0. C. Uhlenbeck (Sampson et al., 1989). The transcripts were purified by high pressure liquid chromatography and the tRNA prepared for “F NMR as described (Chu & Horowitz, 1989). Nuclear magnetic resonance spectra of renatured tRNA samples in sample buffer (50 mv-sodium cacodylate (pH 6.0), 15 mM-MgCl,, 100 mM-NaC1, I.0 mm-EDTA, 10% ‘H,O) were recorded on a Kruker WM-300 pulsed FT NMR spectrometer, as

before (Chu & Horowitz, 1989), at 282 MHz and at either 22°C or 47°C. Ghemical shifts are reported as p.p.m. from free FUra; downfield shifts are indicated as positive numbers. “F homonuclear Overhauser effect (NOE) measurements were carried out at 25°C on (FUra)tRNAv”’ (10 mg), in sample buffer prepared in IOOo/o ‘H,O. The pulse sequence consisted of a 300 ms presaturation pulse (5 mW) centered at the frequency of interest, followed by a 10 ps delay, a 90” observation pulse. and a 45 s relaxation delay. A total of 4000 scans were collected. Presaturation pulses were of sufficient powser to saturate the irradiated peak by 80%. Nuclear Overhauser effects were observed by taking the difference between the onresonance spectrum and a spectrum collected with the presaturation pulse set off-resonance, 4 p.p.m. upfield from the FUra standard peak. Aminoacylation kinetics of wild-type and mutant valine tRNBs were determined, as described (Chu & Horowitz, 1989), using purified VRS (Chu & Horowitz, 1991a).

3. Results and Discussion Assignment of resonances from the four 5fluorouraciE residues involved in tertiary interactions, FU8, FU12, FU54 and FU55 (Fig. I), by the nucleotide-substitution method presents di-ficulties because replacement of these nucleotides disrupts tRNA tertiary structure, resulting in drastically altered 19F NMR spectra. A number of different approaches, including 19F homonuclear NOE, were used to assign these resonances. (a) Assignment 5-Jluorouracils

of resonances corresponding

54 and 55 in the 1 9F NM of (F Ura) tRNA G’

to

R spectrum

Replacement of FU54 with any nucieotide (other than U) yields mutant tRNAs whose I917 NMR spectra are poorly resolved and exhibit extensive peak broadening (data not shown, but see the spectrum of yeast (FUra)tRNAPh” mutant FU54A in Fig. 5B), an indication that the 2” and 3” structure of the tRNA is disrupted by these substitutions. This is not surprising: FU54, hke the conserved ribothymidine at position 54 in yeast tRNAPh” (Quigley & Rich, 1976), undoubtedly forms an intraloop reverse Hoogsteen base-pair with A58 that helps stabilize the conformation of the T-loop. Model building indicates that A54 and C54 can also form tertiary base-pairs with A58, involving: one or two hydrogen bonds (Sampson et al. i 1990). Lead ion cleavage studies of yeast tRNAP”” suggest, however, that tRNAs having a base other than U at position 54 fold improperly (Behlen et al., 1990). Hydrolysis of the phosphodiester backbone at position 17, mediated by a tightly bound, Fb2+ accurately positioned by the tertiary interact’ions of the T and D-loops (Brown et al., 1985: Krzyzosiak et al., 1988), is a useful gauge of tRNA foolding (Behlen et al., 1990). Both the A54 and C54 mut~ants of yeast tRNAPh” show reduced rates of leadi cleavage (Behlen et al., 1990), implying disruption of tertiary structure.

19F NMR

Chemical

Shift and tRNA

Structure

1175

A c C A PO . C G * C G * C 4F -A G . C A. F67

I7 F G j

C -0 C -G 29F. A 6.G C * G C 330 34F~c

A A

Figure 1. Cloverleaf structure of E. coli S-fluorouracilsubstituted tRNA”“’ with uracil and uracil-derived bases replaced by 5-fluorouracil (F).

The interaction between the I> and T-loops is maintained by a tertiary hydrogen bond between the O-2 of the conserved $55 (U55 or FU55 in the in vitro transcript) and the N-2 and/or N-3 of G18, as well as by hydrogen bonding between the imino proton of U55 (FU55) and an oxygen of phosphate 58 (Quigley & Rich, 1976). When cytosine is substituted for U55 (or FU55), the O-2 of C55 can serve as a hydrogen bond acceptor from G18, but hydrogen bonding with the oxygen of P-58 is eliminated. The single hydrogen bond is evidently sufficient for the tRNA to retain a nearly native tertiary structure, as shown by the “F NMR spectrum of the tRNAV”’ mutant FU55C. This closely resembles the spectrum of wild-type tRNAVa’, except’ for resonances A and I (marked by arrows in Fig. 2), one of which is missing due to the replacement of FU55 by C, whereas the other has shifted upfield to a position between peaks M and N, at 2.1 p.p.m. (Fig. 2A). FU55 can, therefore, be assigned to either A or 1. Small upfield shifts of peaks C (FU59) and G (FU7) (assigned by Chu et al., 1992) are also observed (Fig. 2). Substitution of FU55 with a purine (A or G) distorts the phosphodiester backbone of the tRNA; the lgF NMR spectra of the tRNA”“’ mutants FU55A and FU55G show broad, poorly defined resonances, making them useless for peak assignments (results not shown). Because replacing FU55 with C55 affects both of the peaks A and Z in the 19F NMR spectrum tRNA”“’ mutant FU55C, it is likely that the two corresponding FUra residues are located near each other in the tRNA molecule. 19F homonuclear NOE experiments demonstrate that peaks A and Z derive from FU54 and FU55. Distance measurements

CHEMICAL

Figure

SHIFT

(PPM

from

FUra)

2. “F

NMR spectrum of in vitro transcribed 5Auorouracil-substituted tRNA”“’ mutant FU6.E. Spectra were recorded at A, 47°C; and B, 22°C. The spectrum of wild-type tRNA”“’ (light lines) is superimposed on that of each mutant tRP;IA (heavy lines).

made from the crystallographic co-ordinates of yeast tRNArh” show, if we assume a similar structure for E. coli (FUra)tRNAVa’, that only the fluorine atoms at FU54 and FU55 are sufficiently close, 4 to 5 A (1 A = 0.1 nm), to give an appreciable 19F NOE. Such an effect between peaks A and Z is observed when resonance A in the spectrum of wildtype (FUra)tRNA”“‘, recorded in 100% 2Hz0 at room temperature, is irradiated to saturation for 300 milliseconds (Fig. 3). The difference between the spectrum irradiated on-resonance and that irradiated off-resonance reveals a distinct NOE to peak Z (Fig. 3B); a reciprocal NOE from peak Z to A can also be seen (Fig. 3C). Power spillover to resonances neighboring peak I in the spectrum occurs, but the NOE to peak A is clearly visible. Because of the l/r6 distance dependence of the NOE, t,hese results permit assignment of A and Z to FU54 and FC55 (or vice versa), because these are the only two fluorine atoms within 4 A of each other in the crystal structure of tRNA. A previously reported NOE between peaks A and B in the spectrum of

W.-C. Chu et al.

1136

c

CHEMICAL CHEMICAL

SHIFT

(PPM

from

FUra)

Overhauser effects in the Figure 3. igF homonuclear NMR spectrum of 5fluorouracil-substituted tRNA’“i. spectrum of 5-fluorouracil-substituted A, i9F NMR tRNAV”’ preirradiated off-resonance; 3, NOE difference spectrum obtained on selective preirradiation on peak A; C, NOE difference spectrum obtained on selective preirradiation on peak I. Spectra were recorded at 25°C.

Va’ (Hardin et al., 1988) was (FUra)tRNA undoubtedly caused by power spillover; a small apparent NOE from A t’o B is visible in Figure 3B. Note (Fig. 3A) that the spectrum of the tRNA in 2H,0 is quite similar to that in H,O (compare with Fig. 2B), except for an upfield shift of the resonances from FUra residues exposed to solvent (solvent isotope shift (SIS)) due to an H,O-‘H,O effect (Hull & Sykes, 1976). As expected, the largest SIS effects are observed at peaks D (FU17), E (FU47), F (FU33) and H (FU34), which originate from FUra residues in the loops of tRNAVa’ (assigned by Chu et al., 1992). Although the experiments just described relate FU54 and FU55 to peaks A and I in the “F NMR spectrum of (FUra)tRNAV”‘, the resonances cannot be individually assigned on the basis of these results. The r9F NMR spectra of mutant E. coli (FUra)tRNAV”’ with A, G and C in place of FU54

Figure 4. Effect

SHIFT

(PPM from

FUra)

of NaCl

on the “F NMR spectrum of &fluorouracil-substituted tRNAPhe. The transfer RNA was dissolved in buffer lacking NaCl and varying amounts of NaCl were then added. Sodium chloride concentrations were: A, 0 mM; B, 100 mrvr; C, 200 mrvr; and D, 400 mM.

were too ill-defined to be interpretable (see previous discussion); however, the spectrum of the FU54A mutant of yeast (FUra)tRNAPh” is sufficiently wellresolved for assignments to be made (Fig. 5). Assignments of peak A in the “F NMR spectrum of yeast (FUra)tRNAPh” can be related to the assignment of resonance A in the spectrum of E. coli (FUra)tRNA”“‘. We showed that the chemical shift of the resonance farthest downfield (peak A) in the r9F NMR spectra of all FUra-substituted tRNAs examined (E. coli (FUra)tRNAVa’ (Hardin et al., 1986), (FUra)tRNAze’, and (FUra)tRNAy”’ (Hardin et al., 1988)) is uniquely sensitive to changes in ionic strength and magnesium ion concentration. Figure 4 shows that the farthest downfield resonance (peak A) in the rgF NMR spectrum of wild-type yeast (FUra)tRNAPhe exhibits the same behavior, shifting upfield more than 2 p.p.m. with increasing NaCl concentration. These results suggest th,at peak A corresponds to the same FUra residue in all fluorinesubstituted tRNAs, probably one at the position of an invariant nucleotide, and its assignment in the

“F

NMR

Chemical

Shift and tRNA

Structure

1177

trum of (FUra)tRNAPh” -FU54A, allow peak A in the spectrum of (FUra)tRNAPh” to be assigned to FU55. For reasons previously discussed, peak A can then also be assigned to FU55 in the spectrum of (FUra)tRNAV”’ and all other fluorinated tRNAs. Interestingly, Redfield collaborators and (Johnson & Redfield, 1981; Tropp & Redfield, 1981) observed a resonance in the 196 to 199 p.p.m. region of the “H NMR spectra of yeast tRNAPhe and E. coli valine and initiator methionine tRNAs whose chemical shift was also hypersensitive to changes in Mg2+ concentration. On the basis of an observed NOE from the methyl group of T54, this resonance was assigned to the imino proton at N-l of +55 (Tropp & Redfield, 1981). In fluorine-labeled tRNAs, the fluorine of FU55 is located at the position normally occupied by the N(l)-H of $55. Having assigned FU55 to resonance A in the i9F NMR spectrum of E. coli (FUra)tRNAV”“, peak I can be assigned to FU54 on the basis of the reciprocal NOE between A and I (Fig. 3). The large, 1.4 p.p.m~ upfield shift of peak 1 (FU54) in the spectrum of mutant (FUra)tRNAV”‘-FU55C (Fig. 2) indicates that replacing FU55 with cytosine causes a major change in the environment of the fluorine of FU54. This large shift is unlikely to be caused by differences in ring current effects induced in FU54 by stacking on C55 rather than FU55, and is presumably due to a conformational change in the T-loop.

CHEMICAL SHIFT (PPM from FUra) Figure 5. 19F NMR spectra of in vitro transcribed 5-fluorouracil-substituted tRNAPhe recorded at 22°C: A, mutant FU55C; and B, mutant FU54A. The spectrum of wild-type 5-fluorouracil-substituted tRIL‘APh’ (light lines) is superimposed on that of each mutant tRNA (heavy lines).

spectrum of (FUra)tRNAPhe will permit assignment of peak A in the spectrum of E. coli (FUra)tRNAV”‘. We were able to make this assignment by i9F NMR spectra of yeast comparing the mutants having substitutions for (FUra)tRNAPh” FU54 and FU55. The spectrum of yeast (FUra)tRNAPh”-FU54A, in which FU54 is replaced by A, shows a resonance at the chemical shift position expected for peak A (Fig. 5B). Although the upfield portion of the spectrum is not well resolved, the farthest downfield resonance can readily be identified as the counterpart of peak A because it chemical shift position is highly sensitive to ionic strength (results not shown). Peak A, however, is missing from the spectrum of the Phe mutant FU55C, in which cytosine (FUra)tRNA substitutes for FU55 (Fig. 5A); several other resonances in the upfield region of the spectrum of this mutant have also shifted (Fig. 5A). The absence spectrum of the of resonance A in -FU55C and its presence in the spec(FUra)tRNAPh’

(b) Assignment of resonance6 corresponding to 5-jiuorouracil residues 8 and 12 in the 19F NMR spectrum of (FUra)tRNA ‘a1 With FU54 and FU55 assigned, the only two FUra residues remaining unassigned, FL% and FU12, must correspond to the two peaks still unassigned, J and M. The FUl2. A23 base-pair in the D-stem is part of the A9-A23-FU12 tertiary base triple in which A9 forms two hydrogen bonds to A23 in the major groove of the FU12.A23 WatsonCrick base-pair; an additional hydrogen bond occurs between the exocylic NH, of A9 and an oxygen of phosphat’e 23. This base triple is adjacent, to the G46-G22-Cl3 and G45-GIO-C25 triples and helps stabilize the sharp bend between residues 9 and 10 in the P-IO loop of the tRNA. Replacement of the FU12 I A23 base-pair with Cl2. G23 disrupts the tertiary interactions, as shown by the 19F NMR spectrum of the mutant tRNA A9-G23-C12, which exhibits broad, poorly defined peaks that cannot be used to assign F12 (results not shown). The same is true for the (FUra)tRNA”“’ mutant A9G-A23G-FUl2C, even though the base triple G9-G23-Cl2 is found in some tRNAs (Sprinzl et al., 1989), and the mutant yeast tRNAPh”, with G9-G23-Cl2 in place of the wild-type A9-A23-U12, can readily be aminoacylated (Sampson et al., 1990) and has a near-native tertairy structure, as indicated by the high rate of Pb2+ cleavage at U17 (Behlen et al., 1990).

1178

W.-C. Chu et al. The conserved tertiary base-pair between FU8 and Al4 represents a second reverse Hoogsteen interaction in the tRNA. This base-pair is stacked on adjacent tertiary base-pairs in the complex core of the tRNA and helps stabilize the bend between residues 7 and 8 in the P-10 loop. It is, t’herefore, not surprising that substitution of FU8 in (FUraJ)tRNAVa’ by G, A or C leads to a disruption of tRNA 3” structure, as demonstrated by major peak broadening in the 19F N’IMR spectra of these mutant tRNAs (results not shown). Because (of this, it is not possible to assign FU8 directly. However, the only resonance in the spectrum of (FUra~)tRNAV”’ remaining unassigned is M, and this can now be assigned to FU8. (c) Aminoacylation 5-Jluorouracil-substituted

CHEMICAL SHIFT (PPM from FWra) Figure 6. Assignment of 5-fluorouracil at, position 12 of .i-fiuorouracil-substituted tRNA”“‘: “F PvTMR spectra of mutant AK-A23CFU12G recorded at A, 47°C and B; 22°C. The spectrum of wild-type Sfluorouraeil-substi(light lines) is superimposed on that of tuted tRNA”“’ each mutant tRXA (heavy lines).

Several tRNAs, including two initiator tRNAs (E. coli tRNA, Met (Woo et al., 1980) and yeast tRNAyet (Schevitz et al., 1979)) and yeast tRINAASp (Westhof et al., 19853, whose crystal structures have been determined, have a G9-C23-G12 triple in place of A9A23-U12. Crystallographic studies show that replacement of the A9-A23-U12 base triple with G9-C23-Gl2 has little effect on the sugar-phosphate backbone configuration of tRNA (Robertus et al., 1974; Klug et al., 1974). Evidently, both base triples stabilize the tertiary folding of the tRNA molecule well. This is also true for E. coli equally (FUra)tRNAVa’: the A9GA23CFU12G mutant of this tRNA, which lacks FU12, gives a well-defined r9F NMR spectrum clearly showing the absence of peak J (arrows in Fig. 6). Resonance J can, therefore, be assigned to FU12. Peak K, which has been assigned to FU29 in the anticodon stem (Chu et al., 1992), shifts as a result of replacement of the semitriple A9-A23-FU12 with conserved base GS-C23-G12, indicating that this substitution affects anticodon stem structure.

kinetics of mutants of tRNA va’ at position 8

The availability of mutants of tRNAV”’ with nucleotide substitutions at position 8 enables us to examine the role of the conserved U8 in the the tRNA. activity of aminoacylation Synthetase-catalyzed exchange of hydrogen for tritium at carbon 5 of U8 (Schoemaker & Schimmel, 1977) and inactivation of synthetase by &bromouridine (Starzyk et al., 1982), suggested formation of a covalent Michael adduct by nucieophilic attack of a SH group at the active site of the aminoacgl-tRNA synthetase on the 6 position of U8 in the tRNA, as a common intermediate in the aminoacylation reaction, at least in prokaryotes. Nevertheless, no 19F NMR evidence for such an adduet was observed (Chu & Horowitz, 1991a). We have now found that Va’ lacking FU8 (FU8A and mutants of (FUra)tRNA FU8C) retain significant, though reduced, lsevels of valine-accepting activity (Table 1). Although the catalytic efficiency of mutant G8 is low, it has a ~l%XJKXl value only O.3o/0 that of the wild-type (FUra)tRNAVa’ (Table l), mutants A8 and C8 are effective substrates for VRS, having Vm’,,,/Kn, values of @I 1 relative to that, of wild-type (FUra)tRNA”“’ (Table 1). Apparently uracil or modified uracil at position 8 is important but not absolutely eissential for the aminoacylation activity of t,RNAVa’. The major effect of nucleotide substitutions for U8 is on Kll> which is 5 to 9 times higher for the Imutant tRNAs than for wild-type tRNAv”*; V,,, values for these mutants are almost identical with that of Table 1 kinetics of E. coli (FUm)tRNA va’ mutants at position X -Relative Vmar Kill (PM) (Pmol/min per mg) Vm.JKm J6,XlL

Aminoacylation

tRNA”“‘t Wild-type PUS -+ A8 FU8 -+ C8

FU8 -+ G8

1.2 66 IQ.5 21.6

3.8 2.2 37 0.22

t tRNA”“’ concentrations used: wild-type FUAA,

FUSC

and FUSG,

1.5 to 13.3 /m.

3.2 034 0.35 6PO1i

(1) 0.11 @ll PO03

(FUX).

0.3 to 27 PM;

“B’ NMR

Chemical

Shift and tRNA

wild-type (FUra)tRNA”“’ (Table 1). Our results demonstrate that replacement of the conserved U8 (FUS) by A or C, although disrupting the tertiary structure of the tRNA, as shown by the poorly resolved 19F NMR spectra of these mutant tRNAs, has only a small effect on the catalytic turnover number of VRS while reducing the affinity of the tRNA for the synthetase. (d) Relationship magnetic

resonance

between jfuorine-19 chemical

shift

of 5-~uorouracil-substituted transfer RNA

and

nuclear the structure

E. coli valine

The 19F NMR spectrum of E. co&i (FUra)tRNA”“’ provides a set of chemical shift markers that are highly sensitive to changes in the 2” and 3” folding of the tRNA. Moreover, the fluorine reporter groups are spread throughout the tRNA and are sensitive to local structural changes at virtually all regions of the molecule. Complete assignment of the spectrum, summarized in Table 2, permits us to monitor changes in tRNA folding produced by differences in et al., 1986, 1988; solution conditions (Hardin Hardin & Horowitz, 1987), by interaction with proteins (Chu & Horowitz, 1988, 1991a) or other ligands (Gollnick et al., 1986, 1987), or by nucleotide substitution(s) (Chu & Horowitz, 1989; Chu et al., 1992). On the basis of assignments (Table Z), a number of generalizations can be made relating the location of a FUra residue within the folded structure of the native tRNA molecule, i.e. in loops, helical stem or tertiary interactions, and the chemical shift position of the corresponding resonance in the 19F NMR spectrum. The five FUra residues in relatively unstructured loops of the tRNA have been assigned to resonances, C, D, E, F and Ei in the central region of the 19F NMR spectrum of (FUra)tRNA”“‘, 4.4 to 5.9 p.p.m. downfield from the standard, free

of resonance

assignments

Peak?

Chemical shift (47°C) 8.0 6.8 59 46 41 49 4.1 44 3.5 33 23 2-l 2.3 2.0

t 19F peaks 1 Assignment 0 Assignment

2

NMR spectrum E. coli tRNA “I

in the “F

substituted

Assignment

FU55 FU641 FU599 PU 173 FU47§ FU33§ PU7§ F‘u3q FU.54 PU12 FU29g FU67§ FU8 FU4§

are designated as in Fig. 2A. made by Chu & Horowitz (1989) made by Chu et al. (1992).

1179

FUra (=0 p.p.m.) (Chu et al., 1992). These results agree with conclusions reached in earlier thermal denaturation studies and with findings regarding the degree of solvent exposure and chemical reactivity of these FUra residues (Hardin et al., 1986). Disruption of the 2” and 3” structure of (FUra)tRNA”“‘, by heating above 8O”C, results in the collapse of the 19F NMR spectrum to a single broad resonance centered at 47 p.p.m., at a position corresponding to the central region of the native tRNA spectrum (Hardin et al., 1986; Chu & Horowitz, 1991b). Furthermore, poly(FU), which exists as a random coil under the experimental conditions used (Szer & Shugar, 1963; Massoulie et al., 1963), also has a 19F spectrum with a single peak at approximately 4.5 p.p.m. (Hardin et al., 1986). The FUra residues corresponding to peaks D (FU17), E (FU47), P (FU33) and H (FU34) are accessible to titration at pH values between 4.5 and 9 and have pK, values of 7.6, which is close to that, of free 5-Auorouridine. These FUra residues are completely exposed to solvent, as determined by the solvent-induced isotope shift (SIS) seen on transfer of the tRNA from H,O to 2H20, and they readily form an adduct with bisulfite, a reagent reacting preferentially with pyrimidine residues in singlestranded regions. The resonance (peak 6) assigned to FU59, in the T-loop of (FIJra)tRNA”“‘, is shifted more than 1 p.p.m. downfield from the central region of the spectrum where the signals from the other loop region FUra residues are located; the peak width of this resonance is also broader than that of the others (Fig. 1). This may, in part, be due to the T-loop being buried in the tertiary structure of the tRNA molecule (Vlassov et al., 1981; Romby et al., 1985). Several lines of evidence indicate that position 59 is not as accessible as other nucleotides in tRNA loops. Uracil 59 in yeast tRNAPh” is known to be protected from reaction with a variety of chemical

Table Summary

Structure

of 5-jluorouracd

Commenti:

Tertiary T-stem: GC wobble T-loop D-loop Variable loop Anticodon loop Acceptor stem Anticodon Tertiary; reverse Hoogsteen D-stem; part of tertiary Anticodon stem Acceptor stem Tertiary; reverse Hoogsteen Acceptor stem

1180

W.-C. Ghu et al.

reagents specific for pyrimidine residues in singlestranded regions of tRNA (for a review, see Goddard, 1977). FU59 in (FUra)tRNA”“’ fails to react with bisulfite, nor does peak C exhibit a SlS on transfer of the tRNA from H,O to *H,O (Hardin et al., 1986). Resonances assigned to FUras base-paired with A (peaks K (FU29, anticodon stem), peak L (FU67, acceptor stem), peak N (FU4, acceptor stem)) are generally found in the upfield region of the “F spectrum of (FUra)tRNAV”’ (1.8 to 2.8 p.p.m.); even FC12 (peak J), which is base-paired with A23 in the D-stem: but is part of the A9-A23-FU12 triple, gives a peak that resonates in this region of the spectrum. An exception is FU7, which is base-paired with A66 at the base of the acceptor stem. Its signal, peak G, region of the spectrum appears in the central (4.9 p.p.m.), close to resonances from FUras in loop regions, probably because FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix. Quite unexpectedly, we found that the FUra hydrogen-bonded to G in the T-stem of FU64, corresponds to a resonance (FUra)tRNA”“‘, (peak B) shifted 4 to 5 p.p.m. downfield from resonances from FUra paired with A. Similar downfield shifts were observed when four other A. FU basepairs (FU29. A41 in the anticodon stem, and A6. FUB?, FU4. A69 and FU7. A66 in the acceptor stem) were individually converted to G. FU basepairs (Chu & Horowitz, 1989). Differences in the stacking geometry of Watson-Crick (FU . A) and wobble (FU G) base-pairs, because of the different functional groups of FUra involved in these structures; significantly change the environment of the fluorine nucleus in the major groove of the helix, accounting for the large downfield chemical shift of resonances from FU. G base-pairs. Hall et al. (1989) structural rearrangedescribe a Ng2’ -dependent ment of in vitro t’ranscribed, unmodified, yeast tRNAPh”, which results in formation of an additional G‘U base-pair in the low Mg*+ form of the r9F NMR spectra of E. coli tRNAVal, tRNA. show no addirecorded at low Mg2+ concentrations, tional low-field resonance (Chu & Horowitz, 1989), suggesting that no extra G. U base-pair is formed in this tRNA. As for resonances from 5-fluorouracil residues that participate in tertiary interactions, previous discussion showed that the signal from FU55, at the position normally occupied by the conserved rT55, is the farthest downfield peak (peak A) in the “F NMR spectra of all FUra-substituted tRNAs. Two FUra residues involved in reverse Hoogsteen tertiary base-pairs, FU54 (peak I, at 3.5 p.p.m.) and FU8 (peak M, at 2.3 p.p.m.), give resonances whose chemical shift positions differ by more than 1 p.p.m. We might expect that FUra residues involved in similar base-base interactions would have comparable chemical shifts. Sampson et al. (1990), however, indicated that these two base-pairs are situated in parts of the tRNA with different phosphodiester backbone conformations and neighboring bases, and

this undoubtedly accounts for the difference in chemical shift of the signals from these two FUra residues. We have shown in this and the accompanying paper (Chu et al., 1992) that the “F N,MR spectrum is extremely sensitive to changes in local or globular structure of tRNA induced by mutation. Transfer RNAs with nucleotide substitutions that cause disruption of tertiary structure show a general deterioration of the “F NMR spectrum. Mutations that cause only local conformational changes yield tRNAs whose spectra are very similar f,o that of the wild-type, with chemical shift changes only to individual resonances. The rgF NlFlR spectrum provides chemical shift markers that are complementary to the chemical shift markers from the ‘H NMR spectrum of the imino protons. Any significant change in hydrogen-bonding will be reflected in changes in the local or global structure of the tRNA, which in turn will cause changes in the “IF spectrum. Roth sets of markers can provide similar information about the global structure of tRNA but they can report on conformational and electrostatic changes at different sites of the molecule. We are grateful to Vahid Feiz for the preparation of several mutant tRNAs, to Jeff Sampson and Olke Uhlenbeck for providing the recombinant plasmid containing the yeast tRNAPh’ gene, and to Deb /Stowers and Jenny Keim for the synthesis of oligonucleotides in the Nucleic Acid Facility of Towa State University. The investigation was supported by grant GM 46546 from the National Institutes of Health and &SF grant DMB 87-04978. This is journal paper no. 5-14137 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, U.S.A., project no. 2566.

References Behlen, L. S.: Sampson, J. R.. DiRenzo. A. B. & Uhlenbeck; 0. C. (1990). Lead-catalyzed cleavage of Biochrmistsy, 29, yeast tRh’APh’ mutants. 25152523. Brown, R. S., Dewan, J. C. & Mug. A. (1985). Crystallographic and biochemical investigation of the lead(D)-catalyzed hydrolysis of yeast phenylalanine tRNA. Biochemistry, 24, 4785-4801. Chu, W.-C. & Horowitz, J. (198X). Interaction of aminoacyl-tRNA synthetase wit,h S-fluorouracilsubstituted transfer RNA: 19F NMR study. Fed. Amer. Sot. Exp. Biol. 2, A772. Chu, W.-C. & Horowitz, J. (1989). i9F N;MR of Ei-fluorouracil-substituted transfer RNA transcribed i:~ vitro: resonance assignment of fluorouracil-guaninte base pairs. Nucl. Acids Res. 17, 7241-7252. Chu, W.-C. & Horowitz, J. (1991a). Recognition of E. coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study. Biochemistry, 30, I6551663. Chu, W.-C. & Horowitz, J. (1991b). Fluorine-19 N%IR studies of the thermal unfolding of SfluorouracilE. coli valine transfer RNA. FEBS substituted Letters, 295, 1599162. Chu, W.-C., Feiz, V., Derrick, W. B. $ Horowitz, J. (1992). Fluorine-19 nuclear magnetic resonance as a probe of the solution structure of muta,nts of ,5-fluorouraeil-substituted E. coli valine tRNA. J. Mol. Biol. 227, 1164-1172.

19F NMR Chemical Shift and tRNA Goddard, J. P. (1977). The structures and functions of transfer RNA. Progr. Biophys. Mol. Biol. 32, 233-308. Gollnick, P., Hardin, C. C. C Horowitz, J. (1986). Fluorine-19 nuclear magnetic resonance study of codon-anticodon interaction in 5-fluorouracil-substituted E. coli transfer RNAs. Nucl. Acids Res. 14, 4659-4672. Gollnick, P., Hardin, C. C. & Horowitz, J. (1987). “F nuclear magnetic resonance as a probe of anticodon E. co& transfer structure in 5-fluorouracil-substituted RNA. J. Mol. Riot. 197, 571-589. Hall, K. B., Sampson, J. R., Uhlenbeck, 0. C. & Redfield, A. G. (1989). Structure of an unmodified tRNA molecule. Biochemistry, 28, X94-5801. Hardin, C. C. & Horowitz, J. (1987). Mobility of individual 5-fluorouridine residues in &fluorouracil-substituted Escherichia coli valine transfer RNA. J. Mol. Biol. 197, 555-569. Hardin, C. C., Gollnick, P., Kallenbach, N. R., Cohn, M. & Horowitz, J. (1986). Fluorine-19 nuclear magnetic resonance studies of the structure of 5-fluorouracilsubstituted E. coli transfer RNA. Biochemistry, 25. 5699-5709.

Hardin, C. C., Gollnick, P. & Horowitz, J. (1988). Partial assignment of resonances in the “F nuclear magnetic resonance spectra of 5-fluorouracil-substituted transfer RNAs. Biochemistry, 27, 487-495. Hills, D. C., Cotten, M. L. & Horowitz, J. (1983). Isolation and characterization of two 5-fluorouracil-substit,uted E. coli initiator methionine transfer RNAs. Biochemistry, 22, 1113-1122. Hull, W. E. 8: Sykes, B. D. (1976). Fluorine-19 nuclear magnetic resonance study of fluorotyrosine alkaline phosphatase: the influence of zinc on protein structure and a conformational change induced by phosphate binding. Biochemistry, 15, 1535-1546. Johnston, P. D. & Redfield, A. G. (1981). Nuclear magnetic resonance and nuclear Overhauser effect study of yeast phenylalanine transfer ribonucleic acid imino protons. Biochemistry, 20, 1144-1156. Klug, A., Ladner, J. E. & Robertus, T. D. (1974). The structure geometry of co-ordinated base changes in transfer RNA. J. Mol. Biol. 89, 511-516. Kryzosiak, W. J., Marciniec, T., Wiewiorowski, M., Romby, P. Ebel, J. P. & Giegit, R. (1988). Characterization of the lead(H)-induced cleavages in tRNAs in solution and effect of the Y-base removal in yeast tRNAph”. Biochemistry, 27, 5771-5777. Massoulie, J., Michelson, A. M. & Pochon, F. (1963). Polynucleotide analogues. VI. Physical studies on Biochim. 5-substituted pyrimidine polynucleotides. Biophys. Acta, 114, 16-26. Quigley, G. J. & Rich, A. (1976). Structure domains of transfer RNA molecules. Science, 194, 796-806.

Edited

1181

Structure

Robertus, J. D., Ladner, J. E., Finch, J. T., Rhodes, D., Brown, R. S., Clark, B. F. C. & Klug, A. (1974). Structure of yeast phenylalanine tRNA at 3 il resolution. Nature (London), 250, 546-551. Romby, P., Moras, D., Bergdoll, M., Dumas, P., Vlassov, V. V.; Westhof, E., Ebel, J.-P. & Giege, R. (1985). Yeast tRNAASP tertiary structure in solution and areas of interaction of the tRNA with aspartyl-tRNA synthetase: a comparative study of the yeast phenyialanine system by phosphate alkylation experiments with ethylnitrosourea. J. Mol. Biol. 184, 455-471. Sampson, S. R., DiRenzo, A. B., Behlen, L. S. $ Uhlenbeck, 0. C. (1989). Nucleotides in yeast tRNAPh” required for the specific recognition by its cognate synthetase. Science, 243; 1363-1366. Sampson, S. R., DiRenzo, A. B., Behlen, L. S. $ Uhlenbeck, 0. C. (1990). Role of the tertiary nucleotides in the interaction of yeast phenylalanine tRNA Biochemistry, 29, with its cognate synthetase. 2523-2532. Schevitz, R. W., Padjarny, A., Krishnamachari, N., Hughes, J. & Sigler, P. (1979). Crystal structure of a eukaryotie initiator RNA. Nature (London), 278, 188-190. Schoemaker, H. J. P. & Schimmel, P. R. (1977). Effect of aminoacyl transfer RNA synthetase on H-5 exchange of specific pyrimidines in transfer RNAs. Biochemistry,

16; 5454-5460.

Sprinzl, M., Hartmann, T., Weber, J., Blank, J. & Zeidler, R. (1989). Compilation of tRNA sequences and sequences of tRNA genes. Nucl. Acids Res. 17; Rl-R172. Starzyk, R. M., Kootz, S. W. & Schimmel, P. (‘1982). A covalent adduct between the uracil ring and the active site of an aminoacyl tRNA synthetase. Nature (London), 298, 136-140. Szer, W. & Shugar, D. (1963). Preparation of poly+fluorouridylic acid and the properties of halogenated poly-uridylic acids and their complexes with polyadenylic acid. Acta Biochim. Pol. 10, 219-231. Tropp, J. & Redfield, A. G. (1981). Environment of ribothymidine in transfer ribonucleic acid studied by means of nuclear Overhauser effect. Biochemistry, 28, 2133-2140. Vlassov, V. V., Giege, R. & Ebel; J.-P. (1981). Tertiary structure of tRNAs in solution monitored by phosphodiester modification with ethylnitrosourea. Eur. J. Biochem.

119,

52-59.

Westhof, E., Dumas, P. 85 Moras, D. (1985). Crystallographic refinement of yeast aspartie acid transfer RNA. J. Mol. Biol. 184, !19-145. Woo, N. H.; Roe, B. A. & Rich, A. (1980). Three-dimensional structure of E. coli initiator tRNAy. Nature (London), 286, 346-351.

by P. E’. Wright