Proc. Natl. Acad. Sci. USA Vol. 74, No. 11, pp. 4900-4904, November 1977 Biochemistry

Sequence of an oligonucleotide derived from the 3' end of each of the four brome mosaic viral RNAs (aminoacylation/tRNA-like structure) RANJIT DASGUPTA AND PAUL KAESBERG Biophysics Laboratory of the Graduate School and Biochemistry Department of the College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706 Communicated by Heinz Fraenkel-Conrat, August 29, 1977

ABSTRACT A -3'-terminal oligonucleotide fragment, 161 necessary for infectivity of BMV RNA (17). Thus, like their 5' bases long, can be obtained from each of the four brome mosaic ends, the 3' ends of these viral RNAs have a distinctive structure virus BNAs by means of nuclease digestion. Like the four intact and presumably an important, although unknown, function. brome mosaic virus RNAs, each fragment accepts tyrosine in- a reaction catalyzed by wheat germ aminoacyl-tRNA synthetase. We have reported that partial hydrolysis of each of the four The complete nucleotide sequence of the RNA 4 fragment has BMV RNAs with RNase T1 releases a 3'-terminal fragment been determined by use of standard radiochemical methods. about 160 nucleotides long-and that this fragment is virtually Comparative data for the fragments from RNAs 1, 2, and 3 show as efficient in accepting tyrosine as the intact BMV RNAs (18). that-the have nearly the same sequence as the RNA 4 fragment. The present paper reports the complete nucleotide sequence The eight bases adjacent to the 3' terminus of the RNA 4 frag- from RNA 4 and gives data ment are identical in sequence to the eight terminal bases of of the fragment derived indicating tyrosine tRNA from Torula utilis and eleven interior bases are that the fragments from the other three RNAs have nearly the identical in sequence to eleven bases encompassing the anti- same sequence. They are each 161 bases long, and we designate codon region of tyrosine tRNA from Saccharomyces cerevisiae, them Q161 of BMV RNAs 1, 2, 3, and 4. T. utilis, and Escherichia coli. Nevertheless, reasonable base- pairing schemes yield, at best, a distorted cloverleaf secondary MATERIALS AND METHODS structure. Materials. T1, T2, and U2 RNases were obtained from Nucleotide sequence analysis of the extremities of plant viral Calbiochem. Pancreatic ribonuclease, snake venom phospho- nucleic acids has provided substantial information regarding diesterase, and bacterial alkaline phosphatase were obtained their structure and their function as messengers. For example, from Worthington Biochemical Corp. Nuclease P1 was ob- the 5'- termini of some of these RNAs, in common with most tained from Yamasa Shoyu Co., Ltd. (Tokyo, Japan). T4 poly- eukaiyotic messengers, contain the "cap" structure m7GpppGp. nucleotide kinase was obtained from P-L Biochemicals, ['y- (For an extensive review, see ref. 1.) The efficiency of transla- 32P]ATP, at a specific activity of 1500-2000 Ci/mmol, was tion is dependent on the presen'ce of cap (2-4). For brome obtained from Amersham/Searle Corp. Nuclease S1, purified mosaic virus (BMV) RNA 4, the monocistronic messenger for by the method of Vogt (19), was a gift from James E. Dahlberg BMV coat protein, the initiation codon is only 10 nucleotides of the University of Wisconsin. Thin-layer plates (CEL 300 from the capped 5' end and this short sequence, together with DEAE or CEL 300 DEAE/HR-2/15) were purchased from the cap, constitutes an effective ribosome binding site (5).i Macherey-Nagel and Co. The 3' termini of some plant viral RNAs have the unique Preparation of BMV RNA and Fragment Q161. The pro- property among messengers that they can quantitatively accept cedures for growth and radioactive labeling of BMV and iso- an amino acid in a reaction catalyzed by aminoacyl'tRNA lation and fractionation of its RNAs have been described synthetase. Thus, turnip yellow mosaic virus RNA (6) and egg (20-22). Partial digestion of the individual BMV RNAs with plant mosaic virus RNA (7) can be charged with valine; tobacco RNase T1 and subsequent isolation of Q161 by electrophoresis mosaic virus RNA can be charged with histidine (8). Each of on polyacrylamide gels have been described (18). Except where the four BMV RNAs can be charged with tyrosine (9). Some explicitly noted, all our descriptions refer to Q161 isolated picornaviral RNAs can be charged, albeit inefficiently and separately from each of the four RNAs. probably only after they have been fragmented (10, 11). The Sequence Analyses. Complete digestion of 0161 with either property of chargeability implies that these RNAs have a T1 RNase or pancreatic ribonuclease, separation of the resulting tRNA-like structure and possibly also a tRNA-like function (12). oligonucleotides by two-dimensional electrophoresis or by A secondary structure, somewhat like that of tRNA, is com- electrophoresis-homochromatography, and further analysis of patible with the sequences determined for the 3'-end regions these oligonucleotides were according to the methods of Sanger of turnip yellow mosaic virus RNA and possibly egg plant and his colleagues (23, 24). mosaic virus RNA (13, 14). The nucleotide sequence at the 3'- The larger oligonucleotides, especially those rich iii pyri- terminal region of tobacco mosaic virus RNA seems less ame- midines, were also analyzed by the wandering spot procedure nable to folding into a cloverleaf secondary structure (15). as described by Silberklang et al. (13). Oligonucleotides were Although the BMV RNAs can be aminoacylated with tyro- labeled at their 5' ends with 32P by use of [y-32P]ATP and sine, this amino acid is not donated to nascent peptides upon polynucleotide kinase according to the procedures of Simsek in vitro translation However, integrity of the 3' end is et al. (25). (16).> T1 and pancreatic ribonuclease oligonucleotide catalogs were obtained for a variety of large and small pieces of Q161, espe- The costs of publication of this article were defrayed in part by the those To obtain payment of page charges. This article must therefore be hereby marked cially having overlapping sequences. large "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: BMV, brome mosaic virus. 4900 Downloaded by guest on October 1, 2021 Biochemistry: Dasguipta and Kaesberg Proc. Natl. Acad. Sci. USA 74 (1977) 4901

T212@9 OT19 ,S with RNase T1, this fragment gives rise to the 3'-terminal oli- * T21 @T20 P21 P19 + gonucleotide A-C-C-AoH, indicating that 0161 is cleaved from T26 S VP T23 P20OP16 * P15 the 3' end of RNA 4. P14 1PI2 Sequence Analysis of RNase T1 and-Pancreatic Ribonu- T25 clease Oligonucleotides of RNA 4 (161. Fragment 0161 of %T24 T13 *T7 P12 PIO S PIN BMV RNA 4, uniformly labeled with 32p (specific activity 109 dpm/mg of RNA), was digested to completion with RNase T1 T14#p *T6 * * P8 Te T5 *7 and the products were separated and characterized. A two- T16+TI70 dimensional separation by cellulose acetate electrophoresis and T5T * P6 DEAE-cellulose thin-layer homochromatography is shown in ,#WT15 OT4 Fig. 1 left. All products were well resolved except the isomers T9 *T3 TO P5 P4 T12 and T13 and T16 and T17. Separation of similar quality T2 is obtainable by two-dimensional electrophoresis (18). Fig. 1 right illustrates complete separation by two-dimensional electrophoresis of the products of digestion of 0161 by pan- creatic ribonuclease. The sequences of most of the pancreatic *PI ribonuclease and of the short T1 RNase oligomerswere deter- Pi! mined by analysis with the complementary RNase T2 RNase, snake venom phosphodiesterase, and U2 RNase. The oligomers FIG. 1. Autoradiograms of 1161 digests. All oligonucleotides were for which such analyses were not definitive were examined, in analyzed by pancreatic, T1, U2, and snake venom (preceded by al- by the wandering spot procedure; these included kaline phosphatase) ribonucleases and some also by the wandering addition, spot technique. (Left) Two-dimensional fractionation of a complete T17-T26 and P15, P17, P18, and P21. Some typical wandering T1 RNase digest of 1161. The first dimension was electrophoresis on spot analyses, those of oligomers T21, T24, T26, and P18, are cellulose acetate at pH 3.5; the second dimension was homochroma- shown in Fig. 2. Fragment 0161 of BMV RNA 4 was digested tography on a DEAE-cellulose (DEAE/HR 2/15) thin-layer plate. The with a mixture of ribonucleases and the products were analyzed dotted circle marked B indicates the position of the blue dye. Note for the presence of unusual bases. No spotswere detected other that the oligonucleotide A-C-C-AOH (spot T8) is readily identifiable nucleotides A, C, and U. from its unusual position on the fingerprint. The spots T1-T26 and than those of the common G, their relative molar yield are: T1, Gp, 8.4; T2, C-Gp, 1.2; T3, A-Gp, Ordering of Oligonucleotides and the Sequence of RNA 3.8; T4, C-A-Gp, 0.8; T5, A-A-Gp, 0.9; T6, C-A-A-Gp, 1.1; T7, A-C- 4 0161. Fragment Q161 of BMV RNA 4 was partially digested A-C-Gp, 0.7; T8, A-C-C-AOH, 0.8; T9, U-Gp, 4.0; T10, C-A-U-Gp, 1.0; with T1 or pancreatic ribonuclease and the pieces were sepa- Til, U-A-C-C-Gp, 0.8; T12, U-A-C-A-Gp, plus T13, C-A-U-A-Gp, rated by polyacrylamide gel electrophoresis or by cellulose 2.0; T14, A-A-U-Gp, 1.0; T15, U-U-Gp, 1.1; T16, C-U-U-Gp, plus T17, acetate electrophoresis and homochromatography. The purified U-U-C-Gp, 2.0; T18, U-C-U-A-Gp, 1.2; T19, A-A-A-A-A-C-A-C-U- Gp, 0.9; T20, A-A-C-C-C-U-U-A-Gp, 0.9; T21, A-C-C-U-C-U-U-A- products were analyzed as had been 0161 itself. Enough pieces C-A-AGp, 1.0; T22, U-A-A-A-U-C-U-C-U-A-A-A-A-Gp, 1.1; T23, of different chain lengths and with overlapping sequences were C-U-C-U-C-U-U-C-Gp, 0.9; T24, C-C-U-U-U-Gp, 1.1; T25, U-C- obtained to permit ordering of all Ti and pancreatic oligonu- U-U-A-Gp, 0.8; and T26, U-U-A-C-U-C-U-U-U-Gp, 1.0. cleotides except those between residues 70 and 74. We could (Right) Two-dimensional fractionation of a complete pancreatic not distinguish between the sequences C-A-U-G-G-G-C-U- ribonuclease digest ofQ161. The oligonucleotides were separated by U-G-C-A-U-A-Gp and C-A-U-G-C-U-U-G-G-G-C-A-U-A- electrophoresis in the first dimension on cellulose acetate at pH 3.5 since both sequences gave rise to the same products after and in the second dimension on DEAE-cellulose paper in 7% formic Gp, acid. The spots Pl-P21 and their molar yield are: P1, Up, 28.7; P2, a variety of partial digestions with T1 and pancreatic ribonu- Cp, 16.6; P3, A-Cp, 7.5; P4, G-Cp, 5.2; P5, A-Up, 1.9; P6, A-G-Cp, 0.9; clease. This ambiguity was resolved by 32P end-labeling of the P7, A-G-A-Cp, 1.0; P8, A-A-A-Up, 0.9; P9, G-Up, 5.0; P10, G-A-A- Ti partial product corresponding to residues 69-74, followed A-A-A-Cp, 0.7: P11, A-G-Up, 1.2; P12, A-A-G-Up, 1.1; P13, A-G-A- by wandering spot analysis as shown in Fig. 3. A-Up, 1.0; P14, G-G-G-Cp, 0.9; P15, A-A-A-A-G-A-G-A-Cp, 0.9; P16, A-G-G-Up, 1.0; P17, A-A-G-A-G:-Up, 1.2; P18, G-G-A-A-G-A-A-Cp, 161 150 140 0.9; P20, G-G-G-Up, 0.9; P21, A-G-G-G- A -C-A-C-G-C-A-G-A-C-C- U -C-U-U-A-C-A-A-G-A- G- U-G-U-C-U-A-G-G-U- 0.9; P19, G-A-G-A-G-Up, 130 120 110 100 G-Up, 0.5. G -C-C-U-U-U-G-A-G-A- G -U-U-A-C-U-C-U-U-U- G -C-U-C-U-C-U-U-C-G- G 90 80 70 A-A-G-A-A-C-C-C-U- U -A-G-G-G-G-U-U-C-G- U -G-C-A-U-G-G-G-C-U- U -G- overlapping pieces, 0161 was digested with 20 ng of pancreatic 60 50 40 C-A-U-A-G-C-A-A- G -U-C-U-U-A-G-A-A-U- G -C-G-U-A-C-C-G-G-G- U -G-U- ribonuclease or 400 ng of T1 RNase per 200 jig of RNA. The 30 20 10 A-C-A-G-U-U-G- A -A-A-A-A-C-A-C-U-G- U -A-A-A-U-C-U-C-U-A- A -A-A-G- incubation was at 00 for 20 min. To obtain comparatively A-G-A-C-C-A-OH shorter pieces (10-30 nucleotides), Q161 was digested with T1 or pancreatic ribonuclease at an enzyme to substrate ratio of Sequences of 0161 from BMV RNAs 1, 2, and 3. The Ti 1:100. The incubation was at 40 for 10 min. The 0161 fragment and pancreatic ribonuclease maps for Q161 from RNAs 1, 2, was tested for the presence. of abnormal bases by two-dimen- and 3 were identical to those of RNA 4 except as follows: (a) In sional thin-layer chromatography on cellulose as described by 0161 from RNAs 1 and 2, spot 11 (U-A-C-C-Gp) was missing Nishimura (26). Conditions for digesting 0161 with nuclease and was replaced by spots corresponding to the oligomers U-Gp S1 were similar to those of Rushizky and Mozejko (27). and U-C-Gp in RNA 1 and U-Gp and C-C-Gp in RNA 2. (ii) A-Cp (spot P3) was reduced in amount while G-Up (spot P9) RESULTS was increased for RNA 1 and G-Cp (spot P4) was increased for Limited digestion of BMV RNA 4 with T1 RNase and subse- RNA 2. Other possible differences are not precluded, e.g., se- quent fractionation by electrophoresis on polyacrylamide gels quence isomers that migrate identically both in electrophoresis produces one major band and many minor bands corresponding and homochromatography. Because extensive differences of to fragments of various lengths (18). The major band fragment, this kind are unlikely and would, furthermore, be unlikely to designated 0161, is the only one produced quantitatively, and have a bearing on our conclusions, we chose, for the present, it can be obtained readily in pure form. On complete digestion to ignore these possibilities. Downloaded by guest on October 1, 2021 4902 Biochemistry: Dasgupta and Kaesberg Proc. Nati. Acad. Sci. US-A 74 (1977)

c A- U \c c 'C \G GA U \G /Cx ._ _<~~~~~~~I :;".~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Il.!, -~~~~~~~~~~~~~~~~~~~~~~~~~~~-k1 /A \G 0

iA oI* I*C /A c -.B _m* G 4A IA \G 2 .. 3 A) .* FIG. 3. Autoradiogram of a partial nuclease P1 digest on a 5'- A I 32P-labeled RNA fragment obtained by partial T1 RNase digestion of 0161. Partial T1 digestion was done on a mixture of uniformly 32P-labeled 0161 (to aid in locating the bands) and unlabeled 0161 \U and the products were separated on 10% polyacrylamide gels. Cor- .r responding bands were then eluted, labeled with kinase at their 5' end, 4"INAG and purified further by two-dimensional electrophoresis and homo- I._ IA chromatography. 2 IA similar locations with respect to their 3' ends. Thus, 1 24 14 1 jc n 161 A-C-U-G-U-A-A - A-U-C-U...A-G-A-G-A-C-C-A T. utilis Tyr tRNA .. A-C-U-G-* -A-i6A-A-' -C-U ... .A-G-A-G-A-C-C-A 46 36 1 FIG. 2. Autoradiogram of partial snake venom phosphodiesterase digests (Upper left and right) and partial nuclease P1 digests (Lower Other sequence correspondences to Tyr tRNAs are five bases left and right) of 5'-32P-labeled spots T21 (Upper left), T24 (Upper long or less. right), T26 (Lower left), and P18 (Lower right) of Fig. 1. Complete Secondary Structure and Base Pairing. Secondary structure T1 and pancreatic ribonuclease digests of 0161 were labeled with is not obviously revealed by inspection of base sequence. Nev- [,y-32PJATP using polynucleotide kinase and the products were sep- ertheless some inferences can be drawn by considering struc- arated. 5'-32P-Labeled spots were then eluted, partially digested with tures that maximize the number of Watson-Crick base pairs. the above enzymes, and analyzed. The first dimension was electro- phoresis on cellulose acetate, pH 3.5; the second dimension was For RNA 4 Q161, the secondary structure of highest stability homochromatography on thin-layer plates made of either pure according to the rules of Tinoco et al. (29) is-shown in Fig. 4. DEAE-cellulose (CEL 300 DEAE; Upper left and right) or a mixture As indicated in that figure, all but one T1 RNase cleavage points of DEAE-cellulose and cellulose in the ratio 2:15 (CEL 300 DEAE/HR of high susceptibility are located in the regions lacking base 2/15; Lower left and right). The homochromatography mixture used pairs. The single exception is between the C-A bond in positions was a 3% solution of yeast RNA in 7 M urea that had been hydrolyzed for 45 min, prepared according to Barrell (24). The mononueleotide 77-78. A number of essentially equivalent base-pairing schemes of T26 waS partially trapped in the paper wick. exist. For example, the sequence A-A-G-A-G-A in positions 4-9 can pair with U-C-U-C-U-U in positions 103-108. X-ray crys- tallography shows that in tRNAs the classical cloverleaf model In all probability RNA 4 (161 and RNA 3(161 are identical, accurately reflects actual base-pairing (although additional base RNA 2 (161 differs only in that base 46 is G, and RNA 1 1161 interactions exist that are not revealed by cloverleaf folding). differs in that base 46 is G and base 45 is U. For 1161, can a cloverleaf structure be drawn with an accessible Sequence Similarity to tRNAs. The susceptibility of q161 A-C-C-A terminus and a tyrosine anticodon centered on an to aminoacylation with tyrosine suggests a structural similarity "anticodon" loop? Both features are believed to be involved in to tyrosine tRNA. Is this resemblance reflected in the (161 recognition by the charging enzyme (30). With some alternative sequence? Comparison shows that only a limited correspon- folding and with elimination of some marginal base pairs the dence to authentic tyrosine tRNAs exists. The sequence of the secondary structure of Fig. 4 can be converted to the more seven and eight bases adjacent to the 3' terminus of (161 is cloverleaf-like structure shown in Fig. 5. However, we are identical to that of the corresponding bases of Saccharomyces unable to construct a secondary structure of high stability that cerevstsae and Torula utilis tyrosine tRNA, respectively (28). provides an "anticodon" loop centered on 1161 bases 14-24. Also eleven bases in the sequence of (161, residues 14-24, have It is possible, of course, that tertiary interactions, unrevealed the same sequences as eleven bases in the anticodon loop region by cloverleaf base pairs, determine the charging enzyme rec- of S. cerevisiae, T. utilis, and Escherichia coli tyrosine tRNA ognition features. Regardless of detailed knowledge of tertiary except that in these tRNAs, three of the bases are modified. structure, it is to be expected that an enzyme recognition site However, the eleven corresponding bases are not in sequentially would be near the surface of a substrate molecule and thus Downloaded by guest on October 1, 2021 Biochemistry: Dasgupta and Kaesberg Proc. Natl. Acad. Sci. USA 74 (1977) 4903

A1 AC145 UC c 0 U A C U UA A U AGAGA1AA CG *U U UCUCUA 0 UA - UG A it CGu GC CG AU AUGUCACAA28 AU GC UACAGUG >: ~~~~~~~~~~d N GC z AU F> I "k AUX GC G>X AA C AGGUGCCUUU U b ,.,tG U G C C ~~i ~ a C A \ G ,C U AG 161AA 90U CCCAAGA A47 , IPAA J-1d-IsotGGGUUC G 0 20 40 60 80 100 G UGC FRACTION NUMBER GCG FIG. 6. Polyacrylamide gel pattern of RNA 4 Q161 that has been -AU / UAAG digested with nuclease S1. The digest was precipitated with ethanol, G A55 dissolved in buffer, and then applied to a 10% polyacrylamide gel. The GCUU Nk electrophoresis was carried out at 3 mA for 2.5 hr, at which time the GU \ bromophenol dye had moved 10 cm. The gel was then crushed in a CG 1 to the bottom UA Gilson automatic gel crusher. Fraction corresponds UA of the gel. GC CG anticodon-like feature, although not the integrity of the anti- A UA c6don itself, is necessary. This is similar to the situation in some 66 tRNAs in which a hidden break in the anticodon loop does not FIG. 4. Possible secondary structures for RNA 4 Q161 drawn to preclude aminoacylation (30). maximum base-pairing. Preferred sites of action of T1 and pancreatic Digestion of Q161 by Si Nuclease. S1 nuclease preferen- RNases are indicated by solid and broken arrows, respectively. tially cleaves in the anticodon loop of tRNAs (31). It is possible to digest tRNA to yield more than 90% half-molecules, that are might be especially vulnerable to nuclease attack. The U-A stable to 50-fold higher concentration of enzyme (27). We thus bond in the "anticodon" region of the structure in Fig. 5 (po- undertook a study of the digestion of Q161 to ascertain whether sitions 66-65) is the most susceptible pancreatic ribonuclease a similar preferential cleavage site existed. (161 was digested point in Q161. With an enzyme to substrate ratio of 1:10,000, with nuclease S1 under conditions optimal for cleaving tRNAs it was possible to obtain a break only at this point in Ql161, in their anticodon loops. After digestion, the products were separating the molecule into two parts. The isolated fragments fractionated on 10% polyacrylamide gels. Fig. 6 shows an ex- were no longer chargeable. However, a molecule with a hidden periment in which 30,000 cpm of 32P-labeled 2161, together break at this point, with the halves still noncovalently bound, with 50Mg of unlabeled yeast RNA, were digested with 80 units was chargeable (M. Bastin and P. Kaesberg, unpublished ob- of enzyme in 0.3 M NaCl, 0.05 M sodium acetate (pH 5.7) for servations), indicating that the structure on both sides of this 24 hr at 00 in a 50-,gl volume. Three major bands, designated 145 I, II, and III, and several minor bands were observed. The RNA AC from each band was eluted and subjected to electrophoresis and U A UA 118 homochromatography after complete digestion with RNase T1. CG U The bands were identified by comparison of their T1 catalogs UA U A with those of 161. Band I contains all Ti oligonucleotides; it CGU GC CG AU A is undigested 161. Band II contains, in equimolar yield, all Ti AU GC C on the 5' side of T13 GC AU C oligonucleotides oligonucleotide (C-A- AU G UUGCU A U-A-Gp) and is thus a cleavage product encompassing residues C AGGUGCCUUU CG 69-161. Band III contains (i) in equimolar amount all Ti G UA C CG oligonucleotides on the 3' side of oligonucleotide T13 except A UA T8, (ii) T8 in less than equimolar amount, and (iii) small oli- C U^AAUU A CUAAAU 15 gomers, in less than equimolar amount, which we believe are 161 G UC portions of T13 or T8. Band III is thus heterogeneous; it contains G A CAA A AUGUCA A residues 1-68, but residues 1-4 and 63-68 exist only fractionally. U A UACAGU A We believe that minor bands a, b, c, and d represent mixtures 90U CCCAAG G A GGGUUC UU63G of SI cleavage products. Bands a and b contained most T1 G G GG oligonucleotides except C-A-U-A-Gp, A-C-C-AOH, and A-C- U U C- A-C-Gp. Bands c and d also contained most T1 oligonucleotides, G GGC UA AU C U GU G G but those near the 5' and 3' ends of Q1i61 were present in low A CG UC 50 amounts. We conclude that S1 nuclease cleaved preferentially UA G UA near residue 66, the "anticodon" region of Fig. 5. G C C 6 DISCUSSION AU 65 FIG. 5. Slightly modified secondary structure for RNA 4 Q161 Earlier publications and this study have shown that the four drawn to illustrate a similarity to the cloverleaf structure of tRNA. BMV RNAs can be enzymatically aminoacylated with tyrosine Downloaded by guest on October 1, 2021 4904 Biochemistry: Dasgupta and Kaesberg Proc. Nati. Acad. Sci. USA 74 (1977) in a manner similar to that of tyrosine tRNA, that a highly by Grant CA 15613 and Training Grant T32 CA09075 from the Na- susceptible ribonuclease T1 cleavage site exists 161 bases from tional Cancer Institute, Grant AI 01466 and Research Career Award their 3' terminus, and that in each case the 3'-cleavage product AI 21942 from the National Institute of Allergy and Infectious Diseases, (0161) can be aminoacylated. Moreover, 0161 is relatively and by Contract 1633 from the Biological Division of the Energy Re- resistant to nuclease digestion; it is obtained in almost quanti- search and Development Administration. tative yield over a wide range of TI concentrations. The 0161 molecules from each of the four RNAs have nearly the same 1. Shatkin, A. J. (1976) Cell 9,645-653. nucleotide sequence. These facts suggest that the 3' end of the 2. Both, G. W., Banerjee, A. K. & Shatkin, A. J. (1975) Proc. Natl. BMV RNAs has a structural resemblance to tyrosine tRNA, that Acad. Sci. USA 72, 1189-1193. it is a tightly folded structure substantially retaining its con- 3. Rose, J. K. & Lodish, H. F. (1976) Nature 262,32-37. figuration after cleavage, and (from its sequence conservation) 4. Shih, D. S., Dasgupta, R. & Kaesberg, P. (1976) J. Virol. 19, that it plays an important role in the life cycle of the virus. 637-642. It was thus gratifying, at least for a short time, that the two 5. Dasgupta, R., Shih, D. S., Saris, C. & Kaesberg, P. (1975) Nature longest sequence identities among S. cerevisiae, T. utilis, and 256,624-628. E. coli tyrosine tRNA, namely, their acylating terminus and 6. Pinck, M., Yot, P., Chapeville, F. & Duranton, H. M. (1970) (ignoring base their anticodon Nature 226,954-956. modifications) loop region, ex- 7. Pinck, M., Generaux, M. & Duranton, H. (1974) Biochimie 56, isted also in 0161. However, these two regions of sequence were 423-428. separated from each other by 28 bases in the tRNAs but by only 8. Oberg, B. & Philipson, L. (1972) Biochem Biophys Res. Commun. four in 0161 and, equally perversely, no manner of stable 48,927-932. base-pairing could produce a structure resembling a tRNA 9. Hall, T. C., Shih, D. S. & Kaesberg, P. (1972) Biochem. J. 129, anticodon loop and stem. However, folding of Q161 into a 969-976. cloverleaf-like structure provides a stem and loop structure in 10. Salomon, R. & Littauer, U. Z. (1974) Nature 249,32-34. which anticodon AUA (bases 65-67), rather than AUG (bases 11. Lindley, I. J. D. & Stebbing, N. (1977) J. Gen. Virol. 34, 177- 19-21) is centered on a loop, as shown in Fig. 5. Moreover, S1 182. 12. Haenni, A. L., Prochiantz, A., Bernard, 0. & Chapeville, F. (1973) nuclease cleaves preferentially at that site just as it does at the Nature 241, 166-168. anticodon of tRNA. Thus there is an evident region of resem- 13. Silberklang, M., Prochiantz, A., Haenni, A. L. & RajBhandary, blance of tertiary structure of 0161 to tRNA but it does not U. L. (1977) Eur. J. Biochem. 72,465-478. include the 11-base-long region of sequence identity. The sig- 14. Briand, J. P., Richards, K. E., Bouley, J. P., Witz, J. & Hirth, L. nificance of the sequence identity is entirely unexplained. It (1976) Proc. Nati. Acad. Sci. USA 73,737-741. may indicate a sequence recognized by the aminoacylating 15. Guilley, J., Jonard, G. & Hirth, L. (1975) Proc. Natl. Acad. Sci. enzyme, an evolutionary vestige, or coincidence. The problem USA 72,864-868. of recognition of tRNA sites by their aminoacylating enzymes 16. Shih, D. S., Kaesberg, P. & Hall, T. C. (1974) Nature 249, is a complex one and after a variety of studies over a period of 353-355. is 17. Kohl, R. J. & Hall, T. C. (1977) Proc. NatI. Acad. Sci. USA, 74, many years only partially solved. (For a comprehensive re- 2682-2686. view, see ref. 30.) The corresponding problem with chargeable 18. Bastin, M., Dasgupta, R., Hall, T. C. & Kaesberg, P. (1976) J. Mol. viral messenger RNAs is equally formidable and may not be Biol. 103, 737-745. worth pursuing until more is known about the functional sig- 19. Vogt, V. M. (1973) Eur. J. Biochem. 33, 192-200. nificance of the charging. 20. Shih, D. S., Lane, L. C. & Kaesberg, P. (1972) J. Mol. Biol. 64, Our sequence data for 0161 do not provide immediate in- 353-362. sight regarding the function of the noncoding region at the 3' 21. Bastin, M. & Kaesberg, P. (1975) J. Gen. Virol. 26,321-325. end of the BMV RNAs. Whatever that function may be, it has 22. Stubbs, J. D. & Kaesberg, P. (1967) Virology 33,385-397. resulted in an evolutionary constraint that provides nearly 23. Sanger, F., Brownlee, G. G. & Barrell, B. G. (1965) J. Mol. Biol. identical 0161 sequences for the four BMV RNAs even though 13,373-398. the in 24. Barrell, B. G. (1971) in Procedures in Nucleic Acid Research, eds. proteins encoded these RNAs are different. Cantoni, G. L. & Davies, D. R., (Harper and Row, New York), The 0161 regions of the BMV RNAs are not necessarily in- Vol. 2, pp. 751-779. volved in translation and amino acid transfer even though this 25. Simsek, M., Ziegenmeyer, J., Heckmen, J. & RajBhandary, U. is an important function of tRNA. Indeed, there is suggestive L. (1973) Proc. Natl. Acad. Sci. USA 70, 1041-1045. evidence to the contrary. BMV RNA that has been chemically 26. Nishimura, S. (1972) Prog. Nucleic Acid Res. Mol. Biol. 12, modified to preclude acceptance of tyrosine is still fully capable 49-85. of serving as a messenger in vitro (16). Certainly, pertinent in 27. Rushizky, G. W. & Mozejko, J. H. (1977) Anal. Biochem. 77, vivo studies are needed. 562-566. Possibly, the 3' ends play an important role in initiation of 28. Barrell, B. G. & Clark, B. F. C. (1975) HandbookofNucleic Acid viral Sequences (Joynson-Bruvvers Ltd., Oxford). assembly. 29. Tinoco, I., Jr., Borer, P. N., Dengler, B., Levine, M. D., Uhlen- Possibly the structure at the BMV RNA 3' ends is an impor- beck, O., Crothers, D. M. & Gralla, J. (1973) Nature New Biol. tant feature of RNA replication. Elongation factors whose 246,40-41. normal function is in translation are needed for the replication 30. Chambers, R. W. (1971) Prog. Nucleic Acid Res. Mol. Biol. 11, of the RNA of phage QB (32). Tryptophan tRNA serves as a 489-525. primer in the synthesis of Rous sarcoma viral RNA (33). Perhaps 31. Harada, F. & Dahlberg, J. E. (1975) Nucleic Acids Res. 2, also, with viruses such as BMV, structures nominally associated 865-871. with translation have a role in replication. 32. Blumenthal, T., Landers, T. A. & Weber, K. (1972) J. Biol. Chem. 249,5801-5808. We thank Christopher Sempos and Chris Saris for technical assis- 33. Faras, A. J., Dahlberg, J. E., Sawyer, R. C., Harada, F., Taylor, tance, Dr. G. Altman for computer analysis, and Drs. J. E. Dahlberg J. M., Levinson, W. E., Bishop, J. M. & Goodman, H. M. (1974) and D. Zimmern for helpful discussions. This research was supported J. Virol. 13, 1134-1142. Downloaded by guest on October 1, 2021