Isolation and partial characterization of valine transfer RNA with uridine and uridine-derived...

J. Mol. Bid. (1974) 88, 361-312

Isolation and Partial Characterization of Escherichia coli Valine Transfer RNA with Uridine and Uridine-derived

Residues Replaced by 5-Fluorouridine

JACK HOROWITZ, CHINQ-NAN Ou, MOHAMBXAD ISHAQ

Depwtmeni of Bioclwmisty and Bbphysica Iowa State University, Amee, Ia 50010, U.S.A.

JAMES OFEN~AND AND JOAN BIEBBAUM

Department of Biochemkty Roche In&We of Molexukw Biology

Nutky, N.J. 07110, U.S.A.

(Received 23 Januury 1974, and in revised form 26 April 1974)

To examine the role of minor bases in transfer RNA, valine 1 transfer RNA of E&erichkwoliwith themajorityof itsuridineanduridine-derivedresiduesreplaced by fluorouridine; was purified by benzoylated DEAE-w&lose chromatography. 87% replacement of pseudouridine, 96% replacement of ribothymidine, dihydrouridine, 4-thiouridine, and uridine and 74% replacement of the uridine-5- oxyaeetio acid were achieved. 6% of the cytidine was replaced by &fluorozytidine. Comparison of the ultraviolet absorbance-temperature profiles and circular dichroism spectra of normal and analogue-containing tRNA indioated that this extensive base substitution had no major effects on the secondary structure of the molecule. The minor bases do not appear to be essential for proper reoog- nition by synthetase sinoe in the aminoaoylation reaction with partially purified valyl-tRNA synthetase, normal and Mluorouridine-containing valine tRNA had virtually the same K,, 1.6 to 1.6~ lob7 M.

1. Introduction

The uracil analogue S-fluorouridine (FUrd) is known to substitute for uridine (Horowitz & Chargaff, 1969) and a number of other uridine-derived minor bases in tRNA such as pseudouridine and ribothymidine (Horowitz & Huntington, 1967 ; Lowrie & Bergquist, 1968; Johnson et al., 1969; Giege etal., 1969a),5,6dihydrouridine (Kaiser et al., 1969; Kaiser, 1971a), 4-thiouridine (Kaiser, 1969a) and 6-methylamino- methyl-2-thiouridine (Kaiser, 1972). Since the role of the modified bases in the struoture and function of tRNA is not yet understood, examination of the properties of FUrd-substituted tRNA should provide insight into the functional significance of the modified nuoleosides.

Previous studies, with unfractionated tRNA, had shown that while FUrd-containing tRNA was capable of accepting most amino acids as readily as normal tRNA (Horowitz & Huntington, 1967; Lowrie & Bergquist, 1968; Johnson et al., 1969; Kaiser, 1969b; Giege et a.!., 1969b) the rate and extent of aminoaoylation of modified tRNA with several amino acids were reduced (Horowitz & Ramberg, 1972). Similarly,

301

302 J. HOROWITZ ET AL,

a number of FUrd-substituted aminoacyl-tRNAs were found to function normally in polynucleotide or phage RNA-directed incorporation of amino acids into poly- peptides. However, others, most notably lysyl-tRNA, served only poorly as amino acid donors (Lowrie & Bergquist, 1968; Horowitz $ Ramberg, 1972).

The level of replacement of uridine and uridine-derived nucleosidea by FUrd in these earlier studies, usually about 70%, was such that it, was difficult to rule out the possibility that the degree of substitution differed in different tRNAs and accounted for the observed variations in activity. To avoid this difficulty and to permit a more direct examination of the role of minor bases in tRNA structure and function we have isolated a purified tRNA species, tRNArs’, in which over 95% of the uridine and the uridine-derived nucleosides are replaced by FUrd. In this paper we compare some physico-chemical and functional properties of this modified tRNA with those of its normal counterpart. Despite major substitutions of the modified pyrimidine bases, the gross structural properties and amino acid acceptance capacity of tRNAg& differ little from those of native tRNA Va*. The accompanying paper (Ofengand et al., 1974) examines the protein synthetic functions of FUrd-substituted Val- tRNAVal.

2. Materials and Methods (a) FUrd-substituted unfra&ionated tRNA (tRNA,,,J

Eecherichia coli strain B, supplied by Grain Processing, Muscatine, Iowa was grown in a glucose-salts medium (Demerec & Cahn, 1953) in 15-1 fermentora (New Brunswick Scientific Co.) at 37°C. 6-Fluorouracil and thymidine were added, at a final concentration of 25 pg/ml each, when the cells reached exponential growth. After 3 h, the cultures were chilled and the cells harvested by centrifugation, washed twice with 0.01 M-Tris.HCl buffer (pH 7.4) containing 0.01 M-magnesium acetate, frozen and stored at -20°C until used.

To prepare RNA, cells were thawed, suspended in 0.01 M-Tris*HCl buffer (pH 7.4) containing 0.01 M-magnesium metate and 2 pg DNAase/ml, and disrupted by passage through a French Pressure Cell (American Instrument Co., Inc). at 10,000 lb/ins. After centrifugation for 30 min at 10,000 g to remove cell debris, ribosomes were pelleted by ultracentrifugation at 105,000 g and tRNA prepared from the ribosome-free supernatant by the phenol/sodium dodecyl sulfate procedure previously described (Johnson et al., 1969). The tRNA was deacylated by incubation at 37°C in 1.8 an-Tris*HCl buffer (pH 8.0) for 90 min (Sarin BE Zamecnik, 1964) and the tRNA purified from contaminating RNA by chromatography on Sephadex GIOO (Schleich & Goldstein, 1966). Transfer RNA highly substituted with FUrd was obtained by chromatography on DEAE-cellulose, &8 described by Kaiser (19693), to remove tRNA which contained little or none of the analogue. The resulting tRNA,,,* had more than 90% of its uridine residues replaced by FUrd.

(b) Putification of tRNAVal substituted by F Urd (tRNA:“&)

The procedure is essentially that of Krauskopf et al. (1972). 8250 AzGO units (0.56 rmol of valine acceptance) of unfractionated tRNA, enriched in the degree of FUrd substitution as described above, was dissolved in 100 ml of 300 mM-NaCl, 10 mm-sodium acetate (pH 5*5), 10 ma6XgS0, and 10 mm-mercaptoethanol and chromatographed on a 1.5 cm x 39 cm column of BD-cellulose (Schwarz-Bioresearch) equilibrated in the same solvent at 4°C. Transfer RNA was eluted with a linear gradient from O-3 M to l-5 M-Naa (1800 ml total vol.) at 1 ml/min, and the valine activity located by acceptance assay. The early eluting major peak of tRNAp’ (0.36 pmol of valine acceptance) wm pooled and freed of salts by precipitation with ethanol. The tRNA was charged with valine in a suitably scaled-up standard assay mixture. When the plateau of charging was reached, the reaction was stopped by phenol extraction, and concentrated by ethanol precipitation. After desalting

6-FLUOROURIDINE-CONTAININU VALINE tRNA 303

Fm. 1. Chromatographio purification of FUrd-substituted valine tRNA. Chromatography of phenoxyaoetylvalyl-tRNArvr,d wns done as desoribed in Materials and Methods. Flow rata was 04 ml/m& end 2.4~ml frsotions were oolleated and assayed both for aoid-preoipitable radioaotivity (-O-O-) and for absorbanoe at 280 nm (--a--m--). Fraotions 75 to 100 were pooled.

on Sephadex G25 equilibrated in 20 mM-potassium acetate (pH 45), the pooled valyl- tRNA (O-27 rmol) was then derivatized with the phenoxyacetyl group. 0.26 pm01 were recovered and loaded onto a second BD-cellulose column (15 cm x 10 cm) equilibrated in 1 M-NaCl, 10 ma-sodium acetate (pH 5*5), 10 nnu-MgSO,, 10 ma6-mercaptoethanol and 10% (v/v) ethanol at 4’C, and eluted with the same solvent. After the underivatized valyl-tRNA and other tRNAs were eluted, the phenoxyacetylvalyl-tRNA was eluted by a gradient made of 76 ml of the above solution and 75 ml of the above solution modified to contain 25% ethanol (Fig. 1). 86% of the recovered valyl-tRNA was found in this second peak with a spec. act. of 1373 pmol/Azeo. The phenoxyacetylvalyl group was removed by incubation in 1 M-Tris, 0.5 ~-Kc1 (pH 49) for 46 mm at 37°C. Re-assay of the stripped material gave a final spec. act. of 1230 pmol/Aze,-,.

(c) Nuckotide compdh

The nucleotide composition of tRNA”*r and tRNAzti ww determined by the tritium labeling method of Randerath et al. (1972). All transfer RNA samples were dialyzed extensively against distilled water before being subjected to nucleoside analysis. Enzy- matic digestion was carried out for 6 h; all other procedures were as described by Rande- rsth et al. (1972). A fraction of the “H-labeled nucleoside t&alcohol mixture, approx. 3, PUi of 3H, was spotted on a 20 cm x 20 cm cellulose sheet and the derivatives separated by 2dimensional chromatography using acetonitrile/ethyl acetate/n-butanol/isopropanol/ 6 x-ammonia (7 : 2: 1: 1: 2.7, by vol.) as solvent in the fhst dimension and t-amyl alcohol/ methyl ethyl ketone/acetonitrile/ethyl acetate/water/formio acid, spec. gr. I.2 (4: 2 : 15 : 2: 1.6: 0.18, by vol.) in the second. Components were visualized on X-ray film by low- temperature fluorography after exposure for 2 to 4 days. The derivatives were eluted with 2 x-aqueous ammonia or with water, a sample added to 10 ml of scintillation fluid containing 0.3% PPO (w/v) and 26% Triton X114 (v/v) in xylene (Anderson & McClure, 1973) and the samples counted. A blank containing no tRNA wss car&d through the same procedure.

304 J. HOROWITZ ET AL.

Recovery studies using E known mixture of nucleosides and individual tRNA species of known sequences showed that in our hands, the recovery of sll nucleosides wss essentially quantitative except for guanosine, 76% recovery, and ‘I-methyl guanosine, 64% recovery (Horowitz & Ou, 1973, unpublished observations). Base compositions reported were corrected for these recoveries. Quantitative recovery of guanosine was obtained when the time of enzymatic hydrolysis was increased from 6 to 22 h.

(d) Optid murements

Transfer RNA used for optical measurements was freed of Mgz+ and other salts by dialysis at 4°C against 0.01 M-EDTA in 0.05 M-Tris*HCl buffer (pH 7.4), for 6 to 24 h followed by dialysis for 18 h against several changes of the following buffers : (a) 0.01 M-

Tris*HCl (pH 7.4). containing 0.01 M-magnesium acetate; (b) the same buffer containing 0.1 M-N&Cl; or (c) the same buffer containing no further addition.

Temperature-absorbance measurements were msde on a Beckman DU spectrophoto- meter coupled to a Gilford model 2000 absorbance reoorder. Each determination included 2 samples and a solvent blank. The temperature in the sample chamber was regulated by a thermostatically controlled jacket surrounding the chamber, and was measured by means of a thermosensor under the cuvette carriage. The concentration of tRNA was adjusted to give an Aaeo of approx. O-4 to 0.5 at room temperature. During the experiment samples were equilibrated at each temperature until both absorbance and temperature readings remained constant, approximately 20 min. Readings were corrected for solvent expansion at higher temperatures.

Cimular dichmism spectra were recorded at room temperature between 360 and 220 nm on a Jaaco ORD/VF/CD-5 spectmpolarimeter equipped with a Sproul Scientifio 5520-2 CD modification. Above 290 nm, measurements were made in a 1 -em cell ; between 220 and 290 nm, a cuvette with a 0*2-cm pathlength was used. The instrument was calibrated using d-camphorsulfonic acid in water before each run. Results are expressed as molar ellipticities per residue.

Extinction coefficients per mol of residue for tRNAV*’ and tRNAT&, caBo, were determined as described by Blum ei al. (1972). A tRNA solution of measured absorbance was hydrolyzed in 0.3 x-NaGH at 30°C overnight. After neutralization with HCl, the absorbance of the hydrolyzed tRNA was measured. The concentration of the nucleotides and that of the intact tRNA could then be determined from the known extinction coeffioients of each residue (Sober, 1970). The calulatted mean residue extinction coefficient for the sum of the nucleotides was IO-4 x 10” for tRNAVai and 10.3 x lo3 for tRNA$,+

(e) Kinetica of dyl-tramfer RNA fmna&im

Transfer RNA was charged with [14C]valine in a reaction mixture containing 40 mar- Tris*HCl (pH 7*4), 5 mnr-magnesium acetate, 72 m&f-NH,Cl, 2 maa-ATP, 6 mM-mercapto-

ethanol, 4 x 10T6 M-[14C]valine (260 mCi/mmol), all other 19 amino acids (1.25 x lo- 4 M)

and varying amounts of tRNA, as indicated. The mixtures were preincubated at 25°C for 6 min before the ma&ion was initiated by addition of enzyme. Incubation was stopped after 6 min by addition of 5% cold trichlomacetic acid. After 10 min in the cold, the precipitate was collected on Millipore filters, dried and counted in a Packard liquid scintillation spectrometer in a toluene/PPO/POPOP mixture. Incorporation of valine into tRNA under these conditions was linear for 10 min.

Partially purified valyl-tRNA synthetase used in these experiments was prepared from E. wZi B as described by Yaniv & Gms (1969). The enzyme was purified through the step involving chromatography on DEAE-Sephadex.

(f) Mate&& The phenoxyacetic ester of N-hydroxysuccinimide was obtained from SchwarzBio-

research. Snake venom phosphodiesterase (oode VPH) and 2. wli alkaline phosphatase (aode BAPF) were from Worthington Biochemioal Corp. Ribonuclease A (type I-A) was from Sigma Chemical Corp. Tritium-labeled potassium borohydride was Amersham-Searle

6-FLUOROURIDINE-CONTAINING VALINE tRNA 30s

no. TRK293. Thin-layer cellulose sheets on plastic baking and RB54 Royal Blue X- ray film were from Eaetman-Kodak Co. Purified normal E. c&i tRNA”“* (lot no. 70733) was obtained from Boehringer Mtmnheim Corp. and accepted 1040 pmol valine per Aaeo unit.

3. Results

(a) Preparation of 5-jhorouridine-substbstitzlted tRNA””

Valine 1 tRNA was selected for study as B typical amino acid-donating tRNA “al because it is the major isoacceptor tRNA of E. wli 8nd is readily purified both

from veline 2 tRNA and other tRNA species. In addition, it has only the eingle #rd residue in the common sequence, and V&RNA is one of the aminoacyl-tRNAs most stable to chemical hydrolysis (Chousterman et al., 1966). The purification followed standard procedures. A single symmetrical peak was obtained in the second step (Fig. l), with a final specific activity of 1373 pmol of valine accepted per A,,, unit of tRNA.

In the course of these experiments the isoaccepting tRNA profiles of a number of other amino acids were a&o examined. Surprisingly, three peaks of methionine- acceptor activity were found. The first two of these were formylatable (formate: methionine ratios of 0.8 to 1.2 were obtained), but the third peak was not (formate: methionine of 0.04). It is not likely that the additional fMet peak corresponded to the tRNA:““$ species recently described by Egan et al. (1973), in which m7G is replaced by A, since this substitution is not normally found in E. wli B species, and it cannot be the crosslinked species frequently found in tRNA samples and separable chromatogrtlphically (Egan et al., 1973; Petrissant & Favre, 1972) because these FUrd-substituted tRNAs lack 4-thiouridine. No further characterization of these two tRNAfMet species have been carried out yet.

(b) Degree of 5-jluorouridine substitution

Previous work has shown that uridine and the uridine-derived nucleosidea, pseudo&line and ribothymidine (Lowrie & Bergquist, 1968; Johnson et al., 1969), 4-thiouridine (Kaiser, 1969a) and dihydrouridine (Kaiser et al., 1969) can be replaced by FUrd. The results presented in P&e I and Table 1 show that after enrichment for FUrd-containing tRNA and purification of a single valine tRNA species, almost all of the uridine-derived nucleotides were substituted. Approxi- mately 96% of the &dine, dihydrouridine and ribothymidine was substituted. Similar levels of pseudouridine substitution were found. According to the data of Table 1, 87% of the pseudouridme was replaced, end an independent analysis by nucleoside ion-exchange chromatography (Uziel et al., 1968), kindly carried out by Dr Mayo Uziel, showed better than 90% replacement. Uridine-b-oxyacetic acid (Plate I, spot X,) was also replaced by FUrd, though leas readily than the other modified uridine constituents; only 74% substitution being achieved (Table 1). The other nuoleosidea were present in the same pruportions in both tRNAs, except for m7Guo which was reduced in the FUrd-containing tRNA.

Examination of the thin-layer chromatogram of the nucleoside t&alcohols derived from the analogue-containing tRNA (Plate I) revealed an additional nucleoside component adjacent to the adenosine derivative, in the region where mlGuo (not present in tRNAvnl) is normally found. Further charaoterization of this constituent

,I: * .-.-- :, l

8

306 J. HOROWITZ. ET AL.

TABLE]

Base composition of tRNAval and tRNA%‘d as determined by chemical 3H incorporation

Nucleoside Expectedt residues per

76 total

tRNA”*’ residues per

76 total f S.D.

tRNA;,, residues per

76 tOtd & S.D.

Ratio FUrd/control

GS 23 23.6&0.4 23.2*0.2 0.98 C 23 23.7hO.3 22.710.3 0.96 A

9:“,,, 13.7f0.2 13.750.2 1.00

U 94*0*3 0.38&0.02 0.04 6-FUrd - 13.3 0.4 + * 1 1.12*0.07 o~lfJ*o~o2 0.13 hU 1 0~80*0~03 0~06+0~01 0.06 m6U 1 0.77kO.07 0*03* 0.01 0.04 m”G$ 1 1*07~0~01 0.44 f 0.04 0.41 ms A 1 0~50&0*10 0.42 0.07 + 0.84 8oow 1 1.18&0*06 0~31+0.01 0.26 6.FC - - 1.46&0.02

The method of anelysi we8 as described in Meterisk and Methods.

t Yank & Berreu (1969). $ Correated for 76% recovery. 5 Corrected for 64% recovery. 11 4-Thiouridine is reoovered as uridine in the 3H lebeling procedure.

both before and after deemination with nitrous acid, led to its identification as 5fluorocytidine (Ou & Horowitz, unpublished observations). Approximately 60/b of the cytidine in tRNA$& is replaced by 5-fluorocytidine. Kaiser & Kwong (1973) also noted the occurrence of 5-fluorocytidine in mixed tRNA from FUrd-treated E. cdi.

4-Thiouridine was determined by the spectral ratio A33.,/A260 measured in 10 mM-

Tris (pH 7*4), 10 mna-Mga+. The control tRNA ratio was 2.19~ lOTa, which compares favorably with values in the literature (Doctor et al., 1969), while a ratio of 0.19 x lOpa was determined for the tRNAvti F~fti preparation. From these ratios, we calculate that less than 8.7% of the normal amount of 4Srd was present in the FUrd- containing tRNA.

(c) Physical properties

Several physical-chemical properties of normal and FUrd-containing tRNAVal were examined to determine possible differences in structure. The extinction coefficient per mole of residue at 260 nm was measured in the presence of (a) 10 mM- Mg2+, (b) 100 mM-Na+, and (c) buffer only, as described in Materials and Methods. The values were: for tRNAVal, (a) 7000, (b) 7100, (0) 7700, end for tRNA$&, (a) 7400, (b) 7900, and (c) 8300, compared to 10,400 and 10,300, respectively, for completely hydrolyzed samples. While the hypochromicities observed in 10 mM-Mga+ for the two tRNAs were similar, those in 100 mM-Na+ were not. The control tRNA showed about the same hypochromicity in Na + as in Mg2 + while the FUrd-substituted tRNA in Na+ showed less hypochromicity and was more like the Mg2 +-free sample than

S-FLUOROURIDINE-CONTAINING VALINE tRNA 307

the Mg2+-containing one. We interpret this result as an indication that tRNA% is less stable structurally than the control when placed under sub-optimal ionic conditions.

Both normal and F&l-substituted tRNAV** exhibited relatively broad, non- co-operative melting profiles at 260 nm in the absence of Mga + (Fig. 2(a)). The extent of thermal hyperchromicity was only slightly higher (22%) for normal tRNAVs’ than for tRNA$& (20%), and the t, values were 52°C and Sl”C, respectively. Addition of Mga + (0.01 M) to the buffer greatly increased the sharpness of the thermal- denaturation profiles (Fig. 2(b)). The t, values were 82°C and 79°C for normal and FUrd-containing tRNAVa’, respectively, and the extent of hyperchromicity was 30% for both.

1.30- ’ I I I I I I__I 8 I n I I I I

- (a) . . (b)

I.25 -

I 18 1% 1 II 1’ ’ 1 ’ 1 ’ 1 ’ 11 20 40 60 80 100 20 40 60 80 100

Temp.(%)

FIG. 2. Tempereture-mbsorbance profile of tRNA”*’ and tRNAzr4 in the absence (a) end presence (b) of magnesium ions (10-z Y). The prooedure wes as described in Materiels end Methods. tRNAVa’, -O--O-; tRNAgrd, -O--O--.

The circular dichroism spectra of tRNAVal and tRNA%d were also examined (Fig. 3). In the absence of Mg a + (Fig. 3(a)), both spectra show negative bands in the region of 230 to 235 run and strong positive bands with maxima at 267 to 268 run. When Mga + is added (Fig. 3(b)), the latter band shifts slightly toward the blue and increases in intensity. The ellipticity of tRNA& in the 266 to 268 run region is a little greater in both the presence and absence of divalent cations than that of normal tRNAVsl. A second negative band is apparent at wavelengths above 295 nm and the largest differences between normal and FUrd-substituted tRNAval are observed in this region. In both the absence and presence of Mga+ the position of this minimum is shifted 8 to 10 mn toward the red and its intensity reduced in the case of tRNA$k compared to its normal counterpart. The differences are greatest in the absence of Mg2+.

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S-FLUOROURIDINE-CONTAINING VALINE tRNA 309

Normal and FUrd-containing tRNA Val had identical electrophoretic mobilities at pH 8.9 in 10% polyacrylamide gels containing 7 M-urea. Furthermore, in the absence of divalent cations, the rate and extent of hydrolysis of both tRNAs by RNAase T, were essentially the same (data not shown).

(d) Aminoacylation of t RNA& Since the FUrd-containing valine tRNA was purified by virtue of its ability to be

acykted with valine, it clearly was recognizable by its cognate synthetsse. However, to study this interaction in more detail, the kinetics of eminoacylation of normal and FUrd-substituted tRNAval were compared. With either a mixed synthetase preparation or a partially purified valine tRNA synthetase (Yaniv & Gros, 1969), the extent of aminoacylation was found to be the same for both tRNA preparations at any enzyme concentration tested (results not shown). Thus the reduced steady-state level of charging observed at low enzyme concentrations with FUrd-substituted tRNALya (Horowitz & Ramberg, 1972) was not found with tRNA&. Such in- complete aminoacylation has been ascribed to an increased rate of enzymic deacylation (Bonnet & Ebel, 1972). Presumably, substitution of uridine and uridine-derived minor base constituents by FUrd does not increase the rate of this deacylation in the case of Val-tRNA,,,,.

The apparent K, of valine tRNA synthetase (at 25°C) was approximately the same for both tRNAs (Fig. 4), 1.5 to 16x 10-l M. This figure can be compared to the value of 55 x lo-’ M (at 30°C) reported by Yaniv & Gros (1969). The F,,, was about 15% higher with normal tRNAVal than with tRNAz&.

4. Discussion

Valine-specific tRNA isolated from E. coli treated with ELfluorouracil has more than 95% of its uridine replaced by FUrd and is, in addition, largely deficient in the uridine-derived minor base constituents found in tRNAVB1, nemely, pseudouridine, ribothymidine, dihydrouridine, 4-thiouridine and uridine&oxyacetic acid (Plate I and Table 1). It should thus be useful in assessing the biological role of these modified bases in tRNA, and in particular, the functional role of @d in the common sequence GT#CG/A (Ofengand et a.L, 1974).

Our finding, that the FUrd-substituted tRNA had the same kinetics of acylation with valine as the control, shows clearly that none of the bases replaced is essential for synthetase recognition, including the #rd in the GT$CG loop. This result rein- forces our earlier explanation for the loss of aoylating activity upon cyanoethylation of this @d (Siddiqui & Ofengand, 1970,1971) which ascribed the inactivation to a perturbation of tertiary structure by introduction of a cyanoethyl group at a critical juncture, rather than to modification of an essential residue. It is also in agreement with recent reports describing ecylation of tRNAs lacking #rd in this loop (Roberts, 1972; Simsek & RajBhandary, 1972; Simsek et al., 1973).

This is also the first report of the substitution of u&lined-oxyacetic acid by FUrd; not an expected result since other uridine-derived nucleosides modified at position 5 were readily replaced. Uridine-li-oxyacetic acid is located in the first position of the anticodon of tRNAVa’, the wobble position. Replacement of the bulky substituent by fluorine had no effect on the interaction of the tRNA with its synthetase (Fig. 4) in this case although specific anticodon bases of some tRNAs can be important for


0.3 I I I I 0 2 4 6 8 IO

I/[tRNAl IO-’ M-’

FIG. 4. Determination of the apparent K, of velyl-tRNA synthetase for tRNA”*’ and tRNAgr:,,. Aminoaoylatione of the indicated smounts of tRNA were carried out at 26°C as desoribed in Met&& and Methods. Value of the K, were determined from the lea&squares line through the data points in the double reciprooal plots shown. (0) normal tRNAVP’, K, = 1*6x lo-’ M; (a) tRNAgd, K, = 1.6x 10-7 M.

recognition by synthetase (see discussion in Schulman t Goddard, 1973). As examples, one could mention that conversion of Cyd to Urd at the wobble position of E. coli tRNAfMst abolished methionine-acceptor activity (Schulman & Goddard, 1973) while in a sulfur-deficient E. cdi tRNAo’” the absence of a thiol modification in the wobble position has no effect (Agris et al., 1973). Nishimura (1972) has suggested that the presence of uridine-5oxyecetic acid permits tRNA?* and tRNAF to recognize codon triple& ending in Urd. It would be interesting to examine the codon response of tRNAzi& in this connection but this has not yet been done.

Replacement of Urd and other nucleosides by FUrd had little effect on the temperature-absorbance profile or circular dichroism spectrum of tRNAVsl (Figs 2 and 3). While it is diEcult to interpret these optical properties in terms of tRNA structure because of the longer wavelength absorption of the fluorinated base, it seems likely that no major secondary structural differences exist. The decreased hyperchromicity of tRNAzrd in the absence of magnesium compared with normal tRNA (Fig. 2) suggests a possible small loss of secondary structure which is consistent with the findings of Massouli& et al. (1966), who reported a decreased thermal stability (at pH 7) of FUra*Ade base pairs relative to UraeAde base pairs in homopolymer complexes.

Differences in the circular dichroism spectra of normal and FUrd-substituted tRNAVs* in the 295 to 310 nm region also indicate the possibility of small structural modifications as the result of FUrd incorporation. This weak negative band at 295 to 310 nm WM first observed by Sarkar et al. (1967) and is very sensitive to changes in RNA conformation induced by heat deneturation (Sarkar et al., 1967), removal of divalent cations (Robison & Zimmerman, 1971; Reeves et al., 1970) or treatment with urea (Adams et al., 1967). It is not found in single-stranded polynucleotides, but is present in most nucleic acids that have double-stranded regions.

In sum, our results show that replacement of Urd and Urd-derived minor nucleosides in tRNAVal by FUrd has little effect on either the structure of the molecule or on its ability to accept amino acids. The function of V&l-tRNAg& in subsequent

6-FLUOROURIDINE-CONTAINING VALINE tRNA 311

steps of protein synthesis is also not affected by incorporation of the enalogue (Ofengand et al., 1974).

An additional point mey be mentioned. The analogue-substituted tRNA appears by all criteria used in our experiments, and those of Raker (1971b) with unfractionated tRNA, to be no lerger than normal tRNA. Presumably neither the replacement of Urd by FUrd nor the absence of minor base constituenta hes prevented the trimming of tRNA precursor molecules (Robertson et al., 1972) to their mature length.

We thank Karen Lueck for expert technical assistance. This investigation was sup- ported in part by a grant to one of us (J. H.) from the U.S. Publio Health Service, GMO9042.

This is Jourmd paper no. J-7796 of the Iowa Agriculture and Home Eoonomics Experi- ment Station, Ames, Iowa; project no. 1648.

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