Effect of nucleoside modifications on the structure and thermal stability of Escherichia coli valinetRNA

Biochimie (1994) 76, i 192-1204 © Soci6t~ franqaise de biochimie et biologic mol~culaire / Elsevier, Paris

Effect of nucleoside modifications on the structure and thermal stability of Escherichia coil valine tRNA A Kintanar, D Yue, J Horowitz* Department of Biochemistry and Biophysics. Iowa State University, Ames, Iowa 5001 !, USA

(Received 5 May 1994; accepted 17 July 1994)

Summary - - Transfer RNA transcribed in vitro lacks the base modifications found in native tRNA. To understand the effect of base modifications on the structure of tRNA, the downfield region of the ~H NMR spectrum of in vitro transcribed E coil tRNAvat in aqueous phosphate buffer in the presence of excess Mge+ was investigated. The resonances of all imino protons involved in hydrogen bonds in the helical stem regions and in tertiary interactions were assigned using two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) and one-dimensional difference nuclear Overhauser effect (NOE) methods, in addition, some aromatic C2 and C8 proton resonances as well as one amino proton resonance were assigned. The chemical shifts of the assigned resonances of unmodified E coli tRNATM were compared with those of the native tRNA molecule under similar solution conditions. The similarity of the NMR data for unmodified and modified tRNA indicates that the in vitro transcribed tRNA has nearly the same solution structure as the native molecule in the presence of excess Mg2+. The only significant differences were the chemical shifts of resonances corresponding to protons in (or interacting with) bases, indicating the possibility of local structural perturbations. The thermal stability of E coli modified and unmodified tRNA TM in the presence of Mg2+ was also investigated by analyzing the temlgrature dependence of the imino proton spectra. Several tertiary interactions involving modified nucleosides in native E coil tRNA TM are less stable in the absence of base modifications. tH NMR / imino proton resonances / 2D NOESY

Introduction

A relatively large proportion (10-20%) of nucleotide bases in native tRNAs are post-transcriptionally modified. Transfer RNA may be synthesized in vitro using T7 RNA polymerase to transcribe a suitable DNA template [1]. These in vitro transcribed tRNA molecules do not have the base modifications found in native tRNA, yet they are still efficiently aminoacylated by their respective cognate synthetases [1, 2], suggesting that the structure of the in vitro transcript is similar to that of native tRNA. Other evidence indicates, hewever, that the lack of base modifications results in a less stable tRNA molecule. For example, the melting temperatures (Tin) of in vio'o transcribed E coli tRNA v,,I [31, yeast phenylalaaine tRNA [ll and yeast aspartic acid tRNA [41 are lower than the corresponding native tRNA molecules under the same conditions.

*Correspondence and reprints

It is important to characterize the structure of the in vitro transcript and compare it to the stlucture of the corresponding native tRNA in order to elucidate the structural and functional roles of the modified bases in tRNA. Moreover, in vitro transcribed tRNA is an attractive model system for studying tRNA function and its interactions with other biomacromolecules during the steps of protein biosynthesis, inasmuch as it is readily synthesized and purified in large quantities and it is also easy to make variant tRNA molecules with base replacements at specific sites. Clearly, a better understanding of the similarities and differences in the structural characteristics of the in ritro transcribed and native tRNA molecules is required to put these model system studies on a firmer footing. NMR spectroscopy may be used to monitor the structure of biomacromolecules in solution. In the case of tRNA, its relatively large size and inherently poor chemical shift dispersion of the sugar IH N M R resonances has so far hindered the assignment of most of the ~H N M R spectrum, thereby precluding the determination of a high-resolution structural model. Nevertheless, several workers have successfully assi-

1193 gned the bulk of the exchangeable imino proton resonances of several tRNA molecules [5-7] using nuclear Overhauser effect (NOE) methods and references to known tRNA crystal structures [8, 91. The pattern and magnitudes of NOEs observed indicate that tRNA adopts the familiar L-shape structure in solution as is found in the crystalline form, and that all tRNA molecules studied so far have a similar conformation in solution. In addition, the chemical shift of an imino proton resonance is exquisitively sensitive to the immediate environment of that proton. Therefore, the assigned imino proton resonances provide a number of reporters distributed throughout the molecule that can report on any conformational changes in that area of the molecule. Similarly, 19F N M R has been used to study 5-fluorouracil-substituted E coli tRNAVal [10, 11 ] and the assigned fluorine resonances provide additional structural markers. Thus, N M R methods pro-

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vide excellent low-resolution structural information on tRNA. Here, we describe a ~H NMR study comparing the structure of unmodified in vitro transcribed E coli tRNA TM with that of native (modified) E coil valine tRNA at high Mg2+ ion concentration (15 raM). We report the systematic assignment of the irnino proton resonances in the 'H NMR spectrum of the in vitro transcript using one- and two-dimensional NMR methods. The assignments of the imino proton spectrum of native E coil tRNA TM could be transferred directly from the previously published spectrum [6]. Comparison of the ~H NMR spectra of unmodified and modified E coil tRNAVa' obtained at similar solution conditions indicates that the in vitro transcribed E coli tRNA TM has a very similar structure to the native molecule under conditions of excess Mg2+ ion, as evidenced by the similar chemical shifts of most of the assigned imino proton resonances and the similar pattern of observed NOEs. Examination of the temperature dependence of the imino proton spectra of modified and unmodified E coli tRNAVa~ in the presence of excess Mg2+ ion reveals that several tertiary interactions involving modified nucleosides in the native tRNA are weaker in the in vit~w transcript.

A-,4U

C-G U-A 3oC-G 40 C-G C A U .6A ~U C A 35

Fig 1. Schematic model of E coil tRNA TM showing the sequence, secondary structure and tertiary interactions, The modified nucleosides (symbol, position) are: 4-thicu.-'acil (s4U, 8); dihydrouridine (D, 17); undine-5-oxyacetic acid (cmoSU, 34); N6-melahyl adenine (m6A); 7-methylguanosine (mTG, 46); ribothymidine (T, 54); and pseudouridine (W, 55). The in vitro transcribed E coli tRNA TM is expected to have the identical sequence, secondary structure and tertiary interactions except that no bases are modified.

DNA templates for in vimJ transcription by T7 RNA polymerase were derived from the recombinant phagemid pVAL11921, containing the cloned wild-type E coil tRNATM gene linked directly to an upstream bacteriophage T7 promoter, as described previously [2]. Mutations were introduced into the cloned tRNATM gene by oligonucleotide-directed mutagenesis [12]. The oligonucleotides were synthesized using standard solid-phase methods by the Nucleic Acids Facility at Iowa State University. Mutant clones were selected by DNA sequence analysis [13]. The double mutant C30U:G40A, which has the cytosine at position 30 substituted by uracil and the guanine at position 40 substituted by adenine, was also investigated for this report. Transcription reactions were performed as described previously [2] using T7 RNA polymerase prepared by the method of Grodberg and Dunn [14]. The transcript was purified by high performance liquid chromatography (HPLC) as described previously [2]. Purified tRNA was precipitated by addition of ethanol, washed with 70% ethanol and dried. For IH NMR spectroscopy, 5-25 mg of tRNA was dissolved in a minimal amount of buffer containing 11 mM sodium phosphate (pH 7.0), 16.5 mM MgCI2, I 11 mM NaCl and dialyzed against two to three changes of 250 ml of the same buffer. The sample volume was adjusted to 405 lal with dialysis buffer, and 45 pl of D~O was added. Native E coil tRNA TM was purchased from Subriden RNA. NMR samples of native E coil tRNATM were prepared as described above. NMR spectroscopy

All IH NMR spectroscopy was performed at 500 MHz on a Varian Instruments Unity-500 NMR spectrometer. All IH NMR

1194

spectra were acquired at 22°C unless otherwise indicated. The chemical shift reference was H20 which was assumed to resonate at 4,80 ppm. Spectra obtained at other temperatures were also referenced to H20. but were corrected for an upfield peak shift of 5 Hz/°C with increasing temperature. The temperature of the NMR sample was regulated by heated (or cooled) air flow and was measu~d by thermocouple near the sample coil. The temperatures were accurate to :!:0.5°C. One-dimensional NMR spectra were collected w i ~ the H20 resonance suppres~d using the 1-1 spin-echo selective excitation pulse sequence [ 15]. Typical parameter ~ttingg and data processing were as described previously [16l, except the spectra were also sometimes apodized with a shifted sine bell (--35°) to enhance resolution. A typical sample containing -7 mg of tRNA required about 20 rain of data acquisition (several hundred scans) to obtain spectra with good signal-tonoise ratios. One-dimensional NOE experiments were performed with a pulse ~quence consisting of a 100 ms presaturation pulse followed by a jump-return pulse 117] to suppress the H20 resonance. The presaturation pulse was of sufficient power to saturate the irradiated peak by -,,90%. The NOE data were obtained by taking the difference between the spectrum collected with the presaturation pulse centered on the peak of interest and a control spectrum collected with the presaturation pulse set offresonance, well downfield. Typically, 3000 scans were collected at each frequency offset. The delay between the pulses of the jump-return sequence was typically 40 ps to allow good excitation of the downfield imino resonances. Other spectral and data processing parameters were similar to those for the I- I echo one-dimensional tH NMR experiments. The two-dimensional NOESY spectrum [181 was obtained using a jump-return pulse sequence [171 as the read pulse [191. The spectrum was made phase sensitive in the first dimension using the method of States et al [201. The mixing time was 150 ms. The spectrum was collected with 2048 complex points in t,~ and 412 complex points in tl. The sweep width was 12000 Hz in each dimension and the recycle delay was 1.0 s. 192 scans were collected per tt experiment and the 90° pulse width was 10 ps. The data were apodized with 60 ° shifted sine bell in both dimensions. Results

The nucleotide sequence, secondary structure and expected tertiary interactions of E coli tRNA TM are shown in figure 1. This schematic model was derived from the crystal structure of yeast phenylalanine tRNA [8, 9]. Imino protons that are involved in hydrogen bonds in base pairs or tertiary interactions are protected from exchange with solvent [21]. These imino protons are visible in the downfield region of the tH NMR spectrum between 1 ! - ! 5 ppm. Based on the structural model, 28 imino protons are protected front exchange by hydrogen bonding between bases and should be visible in the downfield IH NMR spectrum. Additionally,imino protons that are involved in hydrogen bonds to the backbone might also be expected to contribute to the dowi~field spectrum (downfield of 10 ppm), as might strongly hydrogen bonded amino protons [22].

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Fig 2. Expanded region of the IH NMR spectra of tRNA in H~O (10% D20) with 10 mM sodium phosphate, 100 mM sodium chloride and 15 mM magnesium chloride at 22"C. a. Native E coil tRNA TM. b. E coli tRNA TM transcribed in vitro: peaks C and H have intensity corresponding to three protons; peaks i, K and M each have the intensity of two protons. The downfield region of the tH N M R spectra of native and in vitro transcribed E coil tRNAVal in the presence of excess (15 mM) Mr2+ are shown in figure 2. Both spectra show good resolution comparable with the previously published ~H NMR spectrum of native E coil tRNA TM under similar solution conditions [6]. The spectrum of the in vitro transcript (fig 2b) shows intensity corresponding to - 2 9 proton resonances in the region from 11 to 15 ppm. The 24 resolvable peaks in the spectrum are labelled alphabetically going from downfield to upfield (fig 2b). Peaks with more than one proton intensity are also given additional labels (eg C' and C"). The spectrum of native E coli tRNAVa~ (fig 2a) is nearly identical to that published by Hare et al [6] with only small shifts of two or three resonances. The small shifts are attributable to the slightly different solution conditions of the two samples, there being no excess Mg2÷ in the sample of Hare et al [6]. Thus, their assignments [61 are readily transferred to the spectrum in figure 2a. In contrast, comparison of the N M R spectra of the unmodified and modified E coil tRNAV~ (fig 2) reveals enough differences so as to make it difficult to transfer the imino proton resonance assignments of the native tRNA TM [6] to the ~H N M R spectrum of the in vitro transcript. The *H N M R spectrum of the unmodified tRNA must therefore be assigned independently.

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The assignment strategy for the imino proton resonances of tRNA is well established [6]. The stem regions may be identified by NOEs between imino protons of adjacent base pairs. The identity of the stem resonances is established by identifying spectroscopic markers or 'starting points" along the sequence. in native tRNA molecules, the modified bases often provided these spectroscopic markers, but these are obviously lacking in the i,, vitro transcript. A reliable spectroscopic marker is the G-U base pair in the Tstem. This base pair contains two imino protons in proximity and there should be a strong mutual NOE between their resonances. In the NOESY spectrum (fig 3), the strongest cross-peak is between resonances S and V. These resonances may therefore be assigned to imino protons of residues 50 and 64 which fore1 a G-U base pair, although which corresponds to G50 and which to U64 remains to be determined. Additional spectroscopic markers are provided by WatsonCrick A-U base pairs. These base pairs should show a fairly strong NOE between the imino proton of the uracil and the C2 proton of the adenine. The reso-

nance of the latter proton may be distinguished by its narrow linewidth and its chemical shift in the upfieid range (6.~:-7.8 ppm) of aromatic resonances. These imino proton to C2 proton NOEs are visible in the NOESY spectrum (not shown) and also in one-dimensional NOE experiments (fig 4b, c, f). Note that upon irradiation of peak C, there is a strong NOE to an aromatic peak at -7.5 ppm (fig 4b). This aromatic peak appears rather broad because it is actually two partially resolved C2 proton resonances at 7.48 and 7.50 ppm. This becomes clearly evident when the spectra are processed with resolution enhancement (not shown). Imino proton resonances A, C', C", E and L show strong NOEs to C2 proton resonances and are therefore assigned to Watson-Crick A-U base pairs. The assignment of the acceptor stem starts from peak A which shows weak NOEs to peaks L and T in the NOESY spectrum (fig 3). Peak L shows no other NOEs to imino proton resonances. Peak T has a weak NOE to peak E which is visible at lower contours in the NOESY map and is also seen using one-dimensional NOE methods (fig 4c). Peak E shows a strong NOE to the downfield shoulder of peak H, which in turn has an NOE to peak K (fig 3), Peak K, which has the intensity of two protons, has only one other detectable NOE to peak R. Peak R displays no other NOEs. The NOE connectivities L-A-T-E-H-K-R suggest that these seven resonances correspond to imino protons on a single helical stem. Moreover, imino proton resonances L, A, and E derive from Watson-Crick A-U base pairs. These observations are consistent with the assignment of these seven imino proton resonances to the acceptor stem with peak R corresponding to the G 1 imino proton at the acceptor end (see fig 1). The T stem is assigned starting from the already assigned lone G-U base pair (peaks S and V). In previous ~H NMR studies of imino protons in G-U base pairs, the G imino proton is upfield of the U imino proton [6, 23], so V is assigned to G50 and S to U64. The NOE connectivities Q-V/S-J-N-H' are readily determined from the NOESY spectrum (fig 3) and one-dimensional NOE data (fig 4a, d). The spectroscopic marker provided by the G-U base pair properly orients the T-stem assignments with peak Q corresponding to the imino proton of G49 (see fig l). There is a very strong NOE from the upfield part of peak H (H') to peak C (fig 3). Both peak C and peak H have multiple proton intensity, arid the very strong NOE between these peaks probably corresponds to two NOEs. The NOE between H' (G53) and C indicates that one of the imino proton resonances in peak C probably corresponds to the imino proton in the reverse Hoogsteen base pair between A58 and U54. Based on the structural model, this base pair is expected to be stacked on the end of the T stem, and thus its imino proton resonance should display an

1196 NOE to that of G53 (peak H'). In support of this, there is an NOE from peak C to an aromatic peak at 8.37 ppm (fig 4b). This aromatic resonance is broader than the C2 proton resonances and presumably corresponds to the C8 proton resonance of A58. In a reverse Hoogsteen A-U base pair, the C8 proton of the adenine base is quite close to the imino proton of the paired uracil. The A-U Watson-Crick base pair in the middle of the anticodon stem provides a good starting point for assignment. Three of the five imino proton resonances from A-U Watson-Crick base pairs have already been accounted for (peaks A, E and L). The remaining two are part of peak C (C' and C"). The NOE connectivities M-O-C'-H"-(G) can be traced in the NOESY spectrum (fig 3), although the cross-peak between H" and G is partially observed by the diagonal. These five imino proton resonances may be assigned to the anticodon stem. Because the stem is symmetric with respect to the sequence of base pairs, h,~ ,,,,ver, it is not possible to determine the orientation of the assignment. in the C30U:G40A variant, the sequence symmetry of the anticodon stem is disrupted. Analysis of the imino proton spectrum of this tRNA variant (data not shown) allows unambiguous determination of the orientation of the anticodon stem assignments with peak M corresponding to the G39 imino proton. The remaining imino proton resonance from a Watson-Crick A-U base pair (peak C") provides the starting point for assignment of the D stem. The NOE connectivities M'-D-C"-I can be determined from the NOESY spectrum (fig 3) and one-dimensional NOE data (fig 4b). The connectivity assigns these four imino proton resonances to the D stem. Identification of resonance C" as arising fi'om a Watson-Crick A-U base pair provides the spectroscopic marker to orient the assignments, with peak M' corresponding to the G 10 imino proton. Fig 4. Downfield region of nH NMR one-dimensional dif- ..-> ference NOE spectra of the unmodified tRNA at the same conditions and temperature as in figure 2. a. Peak Q has been saturated and shows an NOE to V. b. Peak C has been saturated and shows NOEs to imino peaks at H, I, O, U and aromatic peaks at 8.37, 7.50 and 7.48 ppm. e, Peak E has been saturated and shows NOEs to imino peaks H and T and an aromatic peak at 7.38 ppm. d, The center of irradiation is peak P but peaks O and Q are also saturated because c f power spillover. The observed NOEs are to peak B from F, to peak C from O, and to peak V from Q. e. Peak X has b~en saturated revealing NOEs to peaks C and U. f. The center of irradiation is peak M but peak L is also saturated by power spillover. The observed NOEs are to peak A from L, to a peak at 7.21 ppm from L, and to peak D from M. Other apparent NOEs that have not been labelled are believed to be artifacts of power spillover or incomplete cancellation, or else could not be unequivocally assigned.

Tertiary interaction assignments There are several imino protons which are probably protected from solvent exchange because of hydrogen bonding in tertiary interactions. One of these, the imino proton of U54 located in the reverse Hoogsteen base pair between A58 and U54 has already been

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1197 Table I. Imino proton assignments in E coli tRNAVa. Peak

A B C C' C" D E F G H H' H" I I' J K K' L M M' N O P Q R S T U V X

Position

14.36 14.31 13.85 13.85 13.85 13.71 13.56 13.36 13.21 13.08 13.08 13.08 12.92 12.92 12.81 12.69 12.69 12.62 12.51 12.51 12.38 12.31 12.22 12.08 12.03 I 1.92 I 1.81 11.76 11.37 9.45

Assignment a

A6U67 USA 14 U54A58 U29A41 UI2A23 CI 1(324 U4A69 A26G44 (227(343 G3C70 G53C61 C28G42 C 13G22 G19C56 C51G63 G2C71 G22G46 U7A66 C31G39 G10C25 G52C62 C30G40 GI5C48 G49C65 GIC72 G50U64 G5C68 G 18U55 G50U64 G 18U55

NOE partners lmino

Aromatic b

L, T I, P H, U H", O I, D M, H" 1", H

7.63 7.77 8.37 7.50 7.48

H" E, K C, N C", G B, C"

7.38 7.00

S, V, N H, R A O D H', J M, C" B V K J, V A, E C, X J, S, Q U, C, H'

7.90 7.21

aAssignment is to the U or G imino proton of a base pair except for peak X, which is the GI8 amino proton. Peak S is assigned to the U64 imino proton and peak V to the G50 imino proton. Peak K' is assigned to the G46 imino proton. Peak U is assigned m the U55 imino proton, bln A-U Watson-Crick base pairs, the aromatic NOE partner is assigned to the adenine C2 proton. In A-U Hoosgteen base pairs (peaks B and C), the aromatic NOE partner is the C8 proton of adenine. In the A-G purine-purine base pair (i~ak F), the aromatic NOB partner is the adenine C2 proton. And in the G22-G46 tertiary interaction (peak K'), the aromatic HOE partner is the G22 C8 proton. assigned (peak C). There is a network of NOEs between peaks C, U and X (fig 4b, e). Peak X is upfield of the normal range of imino protons and is probably an amino proton involved in a hydrogen bond. From the crystal structure of yeast phenylalanine tRNA, one expects NOEs from the resonance of the U54 imino proton to peaks corresponding to the imino proton of U55 and the (hydrogen bonded) amino proton of G 18. The imino proton of U55 participates in a hydrogen bond between N3 of the uracil base and O1 of a backbone phosphate [24] and is likely protected from solvent exchange. Moreover, the corresponding proton has been definitively identified in a IH NMR study of 15N-uracil-substituted E coli methionine tRNA [23]. All of these bases are conserved in E coli tRNAWl and

are expected to adopt a similar structure as yeast phenylalanine tRNA. Peak U is therefore assigned to the imino proton U55 and peak X to the amino proton of Gl8. A regular Hoogsteen base pair is expected to form between U8 and A 14, and to stack on the end of the D stem. The weak NOE (fig 3) between peak B and peak I, assigned to the G22 imino proton in the Dstem, suggests that peak B is the imino proton of U8. The NOE from peak B to a probable C8 proton resonance at 7.77 ppm (not shown) from AI4 are in support of this assignment. There is a weak NOE from peak B to peak P (fig 4d). Inspection of the yeast phenylalanine tRNA structure shows that G 15 fore ~s.a rever~ed Watson-Crick base pair with C48 and is

1198 partially stacked on the U8-AI4 base pair. These four bases ,are conserved in E coli tRNA TM and are expected to adopt a similar structure. Thus peak P probably corresponds to the imino proton of GI 5. Only three hydrogen bonded imino protons remain unassigned to specific resonances. The purine residues A44 and G26 form an unusual base pair in yeast phenylalanine tRNA [8, 9]. The position of these residues in E coli tRNA TM is reversed, with a guanine at position 44 and an adenosine at position 26. Nevertheless, a similar purine-purine base pair is expected to form in tRNAV~ resulting in protection of the G44 imino proton from solvent exchange. This structural arrangement places the imino proton of G44 close to the C2 proton of A26. Upon irradiation of peak F, there is a NOE to a fairly narrow peak at 7.0 ppm (data not shown). The chemical shift and linewidth of this peak are characteristic of an adenine C2 proton. Therefore, peak F is assigned to the imino proton of G44. Residue (346 is predicted to form a base triplet with the Watson-Crick pair G22-C 13 by hydrogen bonding in the major groove. This structural arrangement protects the imino proton of (346 from exchange and puts it close to the C8 proton of G22. There is an NOE from the double peak K to a peak at 7.90 ppm which could be a C8 proton. Thus, K' is tentatively assigned to the imino proton of G46. Residues G19 and C56 arc expected to form a Watson-Crick base pair. This base pair is fairly isolated at the outer comer of the L-shaped tRNA molecule. The imino proton o f this base pair is not within NOE distance of any other imino proton protected from solvent exchange. The only unassigned resonance which is likely to be hydrogen bonded imiJ~o proton is the second resonance in peak I (I') and this is thus assigned to the GI9 imino proton. The assignment of the imino proton spectrum of E coli tRNAVal is summarized in table I. Figure 5 compares chemical shifts of assigned imino proton resonances in the ~H NMR spectra of modified and unmodified E coil tRNA TM obtained at the same solution conditions.

Temperature dependence of imino proton NMR spectra The IH NMR spectra of the unmodified tRNA at high Mg2+ concentration (15 mM free) at four temperatures are shown in figure 6. The spectrum in figure 6A was obtained at the same temperature, 22°C, used for the assignment and it is very similar to figure 2b. The spectrum obtained at 35°C (fig 61)) shows only slight shifts and broadening of a few resonances. At 47°C (fig 6C), there is even more pronounced broadening and some of the resonances have lost considerable

intensity. At 60°C (fig 6d), peaks F, G, a component of peak I, and peak X (G18 mnino) are largely gone. A component of peak C, peak L (UT), peak R (G l), and peak U (U55) have broadened considerably and have lost significant intensity. Peaks A (U67), B (U8), another component of peak C, peaks E (U4), P ((315), S (G50) and V (U64) have broadened more moderately. A component of peak I has moved under peak J, peak C has split into two resolved resonances, peak L has merged with peak M, and peak R ((31) has moved under peak Q. The broadening and loss of intensity of several resonances indicates that the corresponding protons have become more susceptible to exchange with solvent because o f disruption of hydrogen bonding and loss of secondary or tertiary structure. In the case where there are multiple components in a peak, the identity o f the resonances which broaden cannot be readily determined from the one-dimensional spectra. Therefore, selected one-dimensional difference NOE data were obtained at 60°C (not shown). The NOE data indicate that resonances C' (U29) and C" (UI2)

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sity as does peak L (U7). Peak R (G 1) has lost nearly all its intensity and peaks S (U64), U (U55) and V ((350) are slightly broadened. At 70°C (not shown), the spectrum is very similar to that obtained at 60°C. Peaks A (U67), E (U4), S (U64), U (U55) and V (G50) continue to broaden and lose intensity, Peaks G (G43), I' (GI9), L (U7) and R (GI) have lost nearly all intensity at 700C, Peaks C' (U29) and C" (UI2) are starting to broaden and lose intensity and peak M (G39) has lost nearly all its intensity. The identity of peaks broadening and losing intensity was verified by one-dimensional difference NOE data (not shown).

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An NMR study comparing the structure of in vitro transcribed yeast phenylalanine tRNA with that of native yeast phenylalanine tRNA has previously been reported by Hall and co-workers [25]. The resolution of the imino proton spectrum of in vitt~ transcribed E coli tRNAV,' shown here was better than the previously published spectrum of unmodified yeast

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Fig 6. Expanded region of ¿H NMR spectra of unmodified E coil IRNATM obtained at different temperatures. The solution conditions were as in figure 2. a. 22°C. b. 35°C. c. 470C. d. 600C: peaks K and M each have the intensity of two protons; peak H has the intensity of three protons. remain, but resonance C (U54) is largely gone at 00°C. Peak C" is broader than C' at 60°C. Peak I remains (under J) but resonance I' (GI9) is lost. All components in peaks H, K and M remain, The IH NMR spectra of the native E coli tRNA TM at high Mg2+ concentration (15 mM free) at four temperatures are shown in figure 7. The four temperatures selected for figure 7 are the same as those in figure 6 to permit comparison 9f the thermal stability of E coli tRNA val with and without nucleoside modifications. The si)ectrum in figuie 7a was obtained at 22°C and is very similar to figure 2a and to the previously published IH NMR spectra of E coli tRNA TM [6]. The spectrum obtained at 35°C (fig 7b) shows only slig.ht shifts of several resonances and a loss of intensity m peak G (G43). At 47°C (fig 7c), there are additional small shifts, and several more peaks have lost intensity. At 60°C (fig 7(1), peak A (U67) has broadened. Peak E (U4) has shifted under K' ((346) and broadened. Nearly all intensity associated with peak G ((343) is gone. Peak I' (G19) continues to lose inten-

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Fig 7. Expanded region of IH NMR spectra of native E coli tRNAV,i obtained at different temperatures. The solution conditions were as in figure 2. a. 22°C. b. 35°C. e. 47°C. d. 60°C.

1200 phenylalanine tRNA [25]. We can only speculate that the yeast phenylalanine tRNA sample of Hall and coworkers suffered from aggregation resulting in broad IH NMR resonances, as has been observed in other in vitro transcribed RNA molecules [26]. In fact, we have transcribed yeast phenylalanine tRNA and prepared it for NMR studies as described above, and the resulting imino proton NMR spectrum (not shown) had resolution as good as the spectrum of the in vitro transcribed E coli tRNAV~ (fig 2b). Thus, as has been observed by Szewczak et al [26], aggregation of in vitro transcribed RNA molecules is not a general phenomenon. The excellent resolution allowed us to assign the imino proton spectrum of the in vitro transcript in a straightforward fashion using two-dimensional NOESY and one-dimensional difference NOE methods. The NOESY spectrum was quite useful for revealing NOEs between peaks that have similar chemical shifts. These data are generally obscured in one-dimensional difference NOE spectra because of spillover effects. We were able to obtain a two-dimensional NOESY spectrum with a reasonable signal-tonoise ratio and reasonable resolution in about 24 h using a sample containing 25 mg of tRNA.

Structural comparison of modified and unmodO~ed tRNA It is of interest to compare the assignments of the unmodified E coli tRNAVaJ imino proton spectrum with that of the native tRNA [6]. Because the assignments of the tH NMR spectrum of the in vitro transcript were done independently, any chemical shift differences between corresponding resonances of modified and unmodified tRNA may be attributed to real structural differences. The chemical shift comparison is conveniently done using a chemical shift difference plot (fig 5). For this plot, we have transferred the assignments of Hare et al [6] to the imino spectrum of the native tRNA in figure 2a and used the chemical shifts from that spectrum. We see significant chemical shift differences for peaks B, C, K'~ U and X (our labelling scheme). These peaks each correspond to imino protons on modified bases (B, C, K', U) orto an amino proton (X) on a base that is paired to a modified base. Peak B has been assigned to the imino proton of uridiae at the 8 position which in the native tRNA molecule is modified to 4-thiouridine. Peak C has been assigned to the imino proton of uridine at position 54, which in native tRNA is modified to ribothymidine. Peak K' has been assigned to the imino proton of the guanosine at position 46 which is modified to 7methylguanosine in native tRNA. Peak U is assigned to the imino proton of U55 which is modified to pseu-

douridine in native tRNA. In the case of the imino protons on modified bases, we expect the modifications to significantly perturb the electronic distribution in the ring thus accounting for a large part of the chemical shift difference. For example, dethiolation of s4U8 by chemical modification has been shown to result in an upfield 0.6 ppm shift of the resonance [27], and this is indeed about the chemical shift difference observed for this resonance when we compare spectra of the modified and unmodified tRNA. Peak X has been assigned to the G 18 amino proton, which is involved in a hydrogen bond with I355. One might expect that the modification from uridine to pseudouridine at position 55 could affect the hydrogen bond from 0 2 of U55 to the G 18 amino nitrogen. It is well known that the hydrogen bond strength is correlated with chemical shift [28], there being a progressive downfield shift of the hydrogen bonded proton resonance as the strength of the hycirogen bond is increased. Thus, the upfield chemical shift of peak X in unmodified E coli tRNA TM suggests a weaker hydrogen bond between U55 and G IS. This is supported by the temperature dependence of the imino proton spectra of modified and unmodified tRNA TM (vide infra). Conformational differences between the unmodified and modified tRNA molecules could also account for some of the chemical shift differences, although it would be difficult to quantity the relative contribution of these effects. The results do allow us to conclude that if there are any differences in the structures of the modified and unmodified tRNA molecules (at excess Mg2÷ concentration), they are strictly local, ie in the immediate vicinity of the modified bases. The similarity of the chemical shifts of the assigned imino proton resonances in modified and unmodified tRNA (with the exception of the five peaks already noted, all shift differences are less than 0.1 ppm) indicates that under conditions of excess Mg2+ concentration, the global solution structures of modified and unmodified E coli tRNA TM are very similar. The ~H NMR spectrum of the tRNA variant C30U: G40A allowed us to unambiguously determine the orientation of assignments of the anticodon stem. Previously, only indirect secondary chemical shift arguments had been used to orient the assignments in native tRNA TM [6]. Because we can readily compare our assignments of the unmodified tRNA spectrum with those of the native tRNA spectrum, we can establish that the anticodon stem was indeed oriented properly in the latter assignments [6].

Thermal stability of tRNA at high Mg2+ As noted previously, unmodified tRNAs h~v~ iower melting temperature (Tin) than the corre~:ponding

1201 tRNAs containing base modifications even at conditions of high Mg2+ concenwation [ 1, 3, 4]. In the spo~trum of the native tRNA at 60°C (fig 7d), fewer resonances have broadened or lost intensity due to solvent exchange than in the spectrum of the in vitro transcript at the same temperature (fig 6d). Susceptibility of NMR resonances to broadening by solvent exchange indicates that the associated proton is no longer being protected adequately by being involved in a hydrogen bond. The hydrogen bond must be disrupted in order for chemical exchange of the intervening proton to occur [29]. Therefore, at 60°C, specific hydrogen bond interactions in the unmodified tRNA appear to be more disrupted than in the native tRNA, in keeping with the observation that the in vitro transcript has lower thermal stability than the corresponding native tRNA. Because we have imino proton assignments for both the modified and unmodified E coli tRNA val, we can comment on the relative thermal stabilities of different parts of the tRNA molecule. The IH NMR spectra of unmodified E coli tRNAWa at elevated temperatures (fig 6) reveal that resonances corresponding to some of the protons involved in tertiary interactions become much more susceptible to exchange with solvent and broaden significantly with increasing temperature, eg C (U54), U (U55), I' (GI9), F (G44), and X (GI8 amino). The loss of these tertiary marker resonances at 600C indicates that at this temperature, tertiary interactions involving these protons are largely disrupted. Other tertiary interactions appear, however, to be largely intact at 60*(2 as indicated by the continued presence of their associated proton resonances, eg B (US-AI4), K' (G46-G22) and P ((315-C48). These more stable tertiary interactions are those involved in stabilizing the sharp curve made by the D stem (PlO loop). In the case of the native E coli tRNA TM, only the tertiary marker resonance I' (GI9) has lost significant intensity at 60°C, indicating disruption of this tertiary interaction (GI9-C56) between the D and T loops. The tertiary marker resonance U (~P55) is depleted but still largely intact at 70°C, indicating that the corresponding tertiary interaction (G 18-~F55) is still present at this temperature. Other tertiary interactions are still intact at 70°C as indicated by the continued presence of their associated proton resonances, eg B (U8-A 14), C (T54-A58), K' (G46-G22) and P (G15--C48). Peaks B, C, K' and U are all associated with modified nucleoside bases that are involved in tertiary interactions. Peaks B, C and U all show a decreased susceptibility to broadening by solvent exchange in the spectrum of the native tRNA at 60°C as compared to the spectrum of the in vitro transcript at the same temperature. Therefore, we can conclude that base modifications to nucleotides at positions 8, 54 and 55

contribute to the increased thermal stability of the associated tertiary interactions. In general, the resonances of imino protons in Watson-Crick A-U base paJ~ broaden at lower temperatures than those of imino protons in Watson-Crick G-C base pairs, reflec~ng the fact that an A-U base pair is known to be weaker than a G-C base pair. In spectra of the unmodified tRNA at elevated temperature (fig 6), peaks A (U67), C" (UI2), E (U4) and L (U7) are significantly broadened. Of these four resonances, peak L has been broadened most severely indicating that the corresponding A-U base pair (UT-A66) is less thermally stable than the other A-U base pairs. This may be due to the fact that the UT-A66 base pair is at the junction of the T and acceptor stems, and although the base pair is stacked, there may be defects in the stacking or dynamic fluctuations may cause it to become unstacked. Peak C', which has been assigned to the imino proton in the U29-A41 base pair, is still sharp at 60°C. This is probably a reflection of better stacking interactions of this particular A-U base pair with adjacent G-C base pairs near the center of the anticodon stem, thereby increasing the thermal stability of this A-U base pair. The G-U wobble base pair in the T stem, which is associated with peaks S (U64) and V (G50), is also known to be less stable than G-C base pairs. Peaks S and V are indeed broadened, as expected, at elevated temperature and the extent of broadening indicates that the wobble pair is about as thermally stable as AU base pairs U4--A69, U I2-A23 and U67-A6. Based on the broadening of resonances of thermally labile base pairs at elevated temperature, the anticodon stem seems to be the most stable element of the tRNA lacking base modifications, followed by the D and T stems which seem to have about the same stability. The acceptor stem seems to be the least stable element. Peaks A (U67), E (U4), L (UT), S (U64) and V (G50) are broadened to varying degrees in the spectrum of native tRNA at 60°C. Similar to what was observed for the in vitro transcript, peak L is the most severely broadened indicating that the corresponding A-U base pair (U7-A66) is less thermally stable than the other A-U base pairs. Base pairs associated with peaks A, E and S/V are of similar stability in the native tRNA and of about the same stability as the corresponding base pairs in the in vitro transcript. In contrast with the situation for unmodified tRNA, both C' (U29) and C" (U12) are sharp at 60°C, indicating that the associated A-U base pairs are unusually stable. The U12-A23 base pair appears to be more stable in the native tRNA than in the unmodified tRNA. We have shown elsewhere [30] that base modifications stabilize Mg2÷ binding at a site near the D-stem. We have also shown above that the modifi-

1202 cation at U8 contributes to the thermal stability of the US-A 14 tertiary interaction. The improved stability Of the tertiary and metal-binding interactions near the D-stem presumably account for the increased stability of this structural moiety observed in native tRNA. Thus, based on the broadening of resonances of thermally labile base pairs at elevated temperature, the anticodon and D stems seem to be the most stable structural elements in native E coil tRNA TM. The T stem is somewhat less stable and the acceptor stem is the least stable element. The thermal stability of the T stem and the acceptor stem is each about the same in modified and unmodified tRNA. Two G-C imino proton resonances, peaks R (GI-C72) and G (G43-C27), have largely disappeared in the spectra of unmodified tRNA by 60°C (fig 6) indicating that the associated G-C base pairs are thermally unstable. Base pair (GI--C'/2) is at a stem end and because of the absence of stacking interactions on one side, is known to be susceptible to helix fraying [29]. Base pair G43-C27 is ostensibly stacked with a purine-purine base pair G44-A26 (peak F). The absence of peaks F and G at 60°C, however, indicates there is significant disruption of the tRNA structure at the junction of the anticodon and D stems, possibly indicating the presence of some conformational strain. A very similar behavior of peaks R and G was seen in spectra of the native tRNA (fig 7). Peak F was not observed in any NMR spectra of the modified tRNA, but it may be broad and obscured by peak K'. Peak M (G39) arises from an imino proton in a G-C base pair and broadens significantly in the spectrum of native tRNA at 70°C. Base pair C31--G39 is at the junction of the anticodon stem and loop and might also be susceptible to helix fraying. The base pairs at the stem-loop junctions of the D and T stems show no indication of helix fraying at 70°C as indicated by the absence of broadening of their respective resonances. Possibly, the presence of a residual tertiary interaction between the D and T loops stabilizes the D and T stems from helix fraying. It is interesting to compare our results on the temperature dependence of the imino proton spectrum of i~ vitro transcribed E coil tRNA TM with those of Choi and Redfield [31], who studied the thermal stability of uracil imino protons in L~N-labelled native E coil tRNAVat by proton-detected, two-dimensional, heteronuclear (tH-tSN) NMR methods. Their native sample was treated with EDTA, however, and their final buffer contained 1 mM EDTA so the Mg2÷ concentration of their sample was considerably less than in our tRNA samples (15 mM free Mg2+). Choi and Redfield [31] found the uracil resonances in the acceptor stem (U4, U6 and U7) to be thermally iabiie and to exhibit a similar temperature depen-

dence. A uracil in the D stem (U 12) was also found to have a similar temperature dependence as those in the acceptor stem, while one in the anticodon stem (U29) showed little temperature dependence. They also reported little temperature dependence of US0, in the G-U wobble pair: T54, in a tertiary interaction in the T loop; and ~P55, in a tertiary interaction between the T and D loops. Choi and Redfield [31 ] conclude that the tertiary interactions, at least between the T and D loop and within the T loop are more stable than the acceptor stem. Our results on the relative thermal stabilities of A-U base pairs within helical stems are generally in agreement with those of Choi and Redfield [31]. One exception is U7-A66 which, as noted earlier, is markedly less stable than other A-U Watson-Crick base pairs in both the native tRNA and the in vitro transcript. The extreme temperature sensitivity of the U7-A66 base pair has also been noted by Hare et al [6]. In addition, we find that in native tRNA, U 12 in the D stem is as well protected from solvent exchange at elevated temperatures as U29 in the anticodon stem. In contrast to the findings of Choi and Redfield [31 ], we also observe some loss of tertiary interactions between the D and T loops at elevated temperature. This is especially true in the case of the in vitro transcript. In both the modified and unmodified tRNA, however, the tertiary marker resonance r (GI9-C56) loses intensity at a relatively low temperature (47°C), before any broadening of A-U or G-U imino proton resonances is observed. Choi and Redfield [31 ] would not have been able to observe the loss of the G 19--~56 tertiary interaction because they were observing only uracil imino protons. The differences between our data and that of Choi and Redfield [31] may be due in part to differences in solution conditions, especially the Mg'-+ concentration. Comparison to chemical modification and nuclease digestion studies

The effect of base modifications on the solution structure of tRNA has been probed by treatment of the tRNA with various nucleases or chemical modification reagents [3, 4]. In these experiments, the in vitro transcribed and the native tRNA are each treated with the reagent and the reactivity at various sites is probed by gel electrophoresis. Differences in reactivity at particular sites are interpreted in terms of conformational differences of the modified and unmodified tRNA. The major observation of these studies was a weakening of the D-loop/T-loop interactions in unmodified tRNA compared to the corresponding native tRNA. These findings are compatible with our observation that NMR resonances assigned to imino protons involved in the D-loopfl'-Ioop tertiary inter-

1203 actions are more susceptible to solvent exchange at 60°C in the spectrum of the in vitro transcript than in that of native ~ N A . Concluding remarks The assignment of the imino proton NMR spectrum of in vitro transcribed E coli tRNAVat provides excellent spectroscopic reporters of the structure of the stem regions and the presence of tertiary interactions. This study shows that the structure of the stem regions and the nature of the tertiary interactions is remarkably similar in in vitro transcribed and native E coil t R N A ~ under conditions of high Mg 2÷ concentration. The temperature dependence of the imino proton NMR spectra of modified and unmodified tRNA also indicates that base modifications act to stabilize certain tertiary interactions. This seems especially true in the tertiary interactions involving 4-thiouracil at position 8, ribothymidine at position 54 and pseudouridine at position 55. The imino proton NMR spectrum provides no structural reporters for the loop regions, however, since these imino protons exchange too rapidly with the aqueous solvent to be detectable. Fluorine NMR spectra of 5-fluorouracil-substituted tRNA do provide structural markers of the loop and comparison of the 19F NMR spectra of 5-fluorouracil substituted modified and unmodified E coli tRNA TM indicates that at high Mg2÷ concenti'ation, the structures of these molecules are quite similar [2, 10, 11 ]. Together, these NMR studies support the use of unmodified E coli tgNA TM molecules as model systems for biochemical and biophysical studies under conditions of high Mg 2+ concentration. The effect of low Mg 2+ concentration on the structure of in vivo transcribed E coli tRNAVal and the role of modified bases in Mg2÷ binding by tRNA are explored in a separate paper [30]. Acknowledgments Support for this investigation was providedby grantGM 45546 (to JH) from the National Institutes of Health. This is journal paper No J-15612 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, project no 2566. This research benefited from the use of the 500 MHz NMR and Nucleic Acids Biotechnology Instrumentation Facilities at Iowa State University. References I Sampson J, Uhlenbeck OC (1988) Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proc Natl Acad Sci USA 85.1033-1037 2 Chu WCo Horowitz J (1989) t9F NMR of 5-fluorouracii-substituted transfer RNA transcribed in t,itro: resonance assignment of fluorouracil-guanine base i~rs. Nucleic Acids Res 17. 7241-7252

3 Derrick WB. Horowilz J (1993) Probing structural differences between native and ii~ ~ii,o lranscribed Escherichia coli valine transfer RNA: evidence for stable base medifieation-dependentconformers. Nucleic Acids Res 21.49484953 4 Petter'V, Garcia A, Puglisi J, Grosjeen H, Ebei JP, Florentz C, Gieg~ R (1990) Confmmatinn in solution of yeast tl~qAAsP traltSCt~pts deprived of modified nucloetides. Biochimie 72, 73.%744 5 Heersehap A. Mellema JR. Janssen HGJM. Waiters |ALl. Hansnoot CAO. Hilbers CW (1985) lmino-pmt~ resonances of yeast tRNATM studied by two
1204 26 Szewczak AA, White SA, GewiNh DT~ Moore PB (1990) On the use of T7 RNA polymerase transcripts for physical investigation. Nucleic Acids Res 18, 4139-4142 27 Reid mR. Ribeiro NS, Gould G, Robillard, G, Hilbers CW, Shulman RG (1975) Tertiary hydrogen bonds in the solution structure of transfer RNA. PJ~ocNail Acad $ci USA 72, 2049-2053 28 WaSher G. Pardi A, Wtlthrich K (1983) Hydrogen bond length and tH NMR chemical shifts m proteins. J A m Chem $oc 105, 5948--5949

29 g~ochoyan M, Leroy JL, Gu~ron M (1987) Proton exchange and base-pair lifetimes in a deoxy-duplex containing a purine-pyrimidine step and in the duplex of inverse sequence. J Mol Bio1196, 599--609 30 Yue D, Kintanar A, Horowitz J (1994) Nu¢leoside modifications stabilize Mg2÷ binding in Escherichia coil tRNAVal: an imino proton NMR investigation. Biochemistry 33, 8905-8911 31 Choi BS, Redlield AG (! 992) NMR study of nitrogen- 15 labeled Escher/c/6a coli valine transfer RNA. Biochemistry 31.12799-12802