Nucleic Acids Research

Volume 10 Number 5 1982 Nucleic Acids Research

The nucleotide sequence of the maize and spinach chloroplast isoleucine transfer RNA encoded in the 16S to 23S rDNA spacer

P.Guillemaut and J.H.Weil

Institut de Biologie Mole'culaire et Cellulaire, Universite' Louis Pasteur, 15, rue Descartes, 67084 Strasbourg Cedex, France

Received 16 November 1981; Revised 19 January 1982; Accepted 29 January 1982

ABSTRACT The sequence of maize chioroplast tRNA2 e, encoded in the 16S to 235 rDNA spacer, was determined using in vitro labeling techniques. The sequence is: pG-G-G-C-U-A-U-U-A-G-C-U-C-A-G-U-Gmn-G-D-A-G-A-G-C-m2G-C-G-C-C-C-C-U- G-A-U-t6A-A-G-G-G-C-G-A GnGapUCUCUGGTF---G-U-C-C-A-G-G-A- U-G-G-C-C-C-A-C-C-AOH. This sequence is identical to that predicted from the corresponding gene sequence, after excision of a long intervening sequence (1), but shows the post-transcriptional modifications of this tRNA. Furthermore it demonstrates that the excision of the intron occurs after the second base following the anticodon and that this gene, which is over 1000 base-pair long, is transcribed and processed into a mature functional chloroplast tRNA. The sequence of maize (a monocot) and spinach (a dicot) tRNATIe are shown to be identical. 2

INTRODUCTION A few chloroplast tRNAs have already been sequenced namely Euglena, bean and spinach chloroplast tRNAsPhe (2-4), bean and spinach chloroplast tRNAsLeu (4, 5), bean, spinach and Scenedesmus chloroplast initiator and elongator tRNAsMet (6-10), spinach chloroplast tRNAThr (11), tRNAVal (12) and tRNATrp (13). A great interest has focused recently on tRNAAla and tRNAIle, as their genes have been localized, in E.coli as well as in chloroplasts, in the spacer between the 16S and 23S rRNA genes: In E.coli, which has seven rDNA units, three units contain a gene for tRNAAla and a gene for tRNAIle in the spacer, while four units have a tRNAGlu in the spacer (14-21). Hybridization of total chloroplast 4S RNA suggested the presence of tRNA genes in the spacer of Euglena gracilis (22-24) and Chlamydomonas reinhardii (25, 26) chloroplast DNA. Hybridization studies performed with individual purified Euglena chloroplast tRNAs and restriction fragments of Euglena chloroplast DNA have shown that the spacer region contains the genes for two tRNAs, namely tRNAAla and tRNAIle (27). In higher plants, hybridization to the spacer region has been shown for

©) IRL Press Limited, 1 Falconberg Court, London W1V 5FG, U.K. 1653 0305-1048/82/1005-1653$2.00/0 Nucleic Acids Research

tRNAIle in the case of spinach chloroplasts (28) and for both tRNAAla and tRNAIle in the case of maize and bean chloroplasts (unpublished). Recently DNA sequencing studies have shown the presence of a tRNAAla gene and a tRNAIle gene in the spacers of Euglena (29, 30) and maize chloroplast DNA (1). In the case of maize (1) it was shown that the tRNAIle gene contains a 949 base-pair long intron, and that the tRNAAla gene also contains a long intron which consists of 806 base-pairs. The points where the excision of such an intron takes place could only be postulated (1). Furthermore there was no direct evidence that such a large gene (over 1000 base-pair long) is transcribed and correctly processed into a mature functional tRNA. To answer these questions, we have determined the sequence of maize chloroplast tRNAIle,2' which was shown to hybridize to the spacer (unpublished). We have also determined the sequence of spinach chloroplast tRNAIle2' which was shown to hybridize to the spacer (28), in order to compare the sequence of this tRNA in the chloroplasts of a monocot and a dicot. In the case of spinach, no DNA sequence information is available and it is not known if the tRNAIle gene contains any intron.

MATERIALS AND METHODS Purification of tRNAIle from maize and spinach chloroplasts Crude tRNA, extracted from frozen maize or spinach leaves using standard procedures (31), was fractionated on a BD-cellulose column at pH 7.4 (using a linear NaCl gradient from 0.35 to 1.35 M). Maize or spinach chloroplast tRNAIle2 was identified by aminoacylation with an E.coli enzyme extract. The fractions containing isoleucine accepting activity were concentrated and tRNAIle was then purified from these fractions by two-dimensional polyacrylamide gel electrophoresis (32-34). Sequencing techniques The nucleotide sequences of maize and spinach chloroplast tRNAIle2 were determined using post-labeling methods (for a review see ref. 35). Two appro- aches were used: 1) After treatment of purified tRNAIle2 with venom phosphodiesterase (Worthington), the tRNA was labeled using a-I32PIATP3 (300 Ci/mmole ;Amersham) in the presence of tRNA nucleotidyl transferase ; read-off sequencing gels (36) and mobility-shift analysis (37) were used to analyse the 3' end-labeled tRNA.

1654 Nucleic Acids Research

2) After partial hydrolysis of purified tRNAIle2 with hot water (6), the fragments were labeled at their 5' end using standard conditions (36) and fractionated on a thin 15% polyacrylamide slab gel (38) ; the 5' terminal nucleotide of each fragment was determined after P1 nuclease digestion upon mono- (fig. 1) or two-dimensional thin-layer chromatography performed in the presence of markers (35, 39). This approach, based on the principle of the Stanley and Vassilenko method (40), allows to distinguish between the two major pyrimidines and to identify the modified nucleotides ; furthermore, a few fragments eluted from the polyacrylamide gel were subjected to mobility- shift analysis (37) in order to confirm the sequence of specific regions of the tRNA molecule.

RESULTS AND DISCUSSION The data obtained from read-off sequencing gels, from mobility-shift analysis and using the Stanley and Vassilenko technique, allowed to determine the complete sequence of maize and spinach chloroplast tRNAsIle which are 2

T0 C K) U C A G U GmG D A G A G C ,GG C C C C U G

A U tA A G G G C G A Gm7G.4UC U C U G G T v c Fig. 1. Sequence analysis of maize chloroplast tRNA«le using the Stanley and Vassilenko technique (position 12 to 56).Fragments eluted from the polyacrylamide gel were totally digested with P1 nuclease. The 5'termini were separated on cellulose plates (20 x 20 cm) using the following solvent: 2-pro- panol :conc.HCl :H20, 70:15:15 (v/v/v)

1655 Nucleic Acids Research

identical except for the percentage of methylation of the G at position 46 (fig. 2). Maize and spinach chloroplast tRNAsIle26are both 75 nucleotide long. They have the usual T* sequence in loop IV, a D and a Gm in loop I, and a t6A adjacent to the anticodon. At position 26 there is a m2G, which has so far mainly been found in eucaryotic tRNAs, but is also present at this position in Scenedesmus obZiquus chloroplast initiator tRNAMet (8) in yeast mitochondrial tRNAPhe (41) and N.crassa mitochondrial tRNA yr (42). The modified nucleotide acp3U at position 47 has also been found at this position in bean and spinach chloroplast tRNAsPhe (3-4), in bean chloroplast initiator tRNAFF t (6) and in a number of E.coli tRNAs (43). The anticodon GAU allows chloroplast tRNAIle to read the isoleucine codons 2 Ile AUC and AUU. But in maize and spinach chloroplasts there is another tRNA called tRNAIle (33, 34), which has not been studied, except for mapping the corresponding gene on chloroplast DNA (33), and is perhaps able to read the third isoleucine codeword (AUA). The sequence of maize chloroplast tRNAIle2 is identical to that predicted by Koch et al. (1) from the corresponding gene sequence, but shows the post- transcriptional modifications of this tRNA. Koch et al. (1) have studied the sequence of the maize chloroplast spacer located between the 16S and the 23S rRNA genes and have shown that it contains a tRNAIle gene interrupted by a

A C C A pG - C G - C 70 C - G U - G A- u 60 U - A o U GA 5U GGACC A G A 1C A I I A U C UC G UC5U GG C Gm IiC U T I$ G G AG D A WIG-

C - G 30 C G-40 soCC-G- G-4 C A u e6A G A U :is Fig. 2. Nucleotide sequence of maize and spinach chloroplast tRNAsPle (In spinachi chloroplasttRNAIle G at position 4b is only partially methylated into m7G).

1656 Nucleic Acids Research

949 base-pair intron. They have postulated three putative splicing points, namely i) immediately after the anticodon (on its 3' side), ii) after the first base following the anticodon, iii) after the second base following the anticodon. Depending upon where the splicing occurs, the anticodon loop of the mature tRNA should have the following sequence: i) GAUAC, ii) GAUAC or iii) GAUAA, respectively. Our results (fig. 1) show that the anticodon is followed by two A residues (the first of which is modified into t6A) and thus demonstrate that the third hypothesis is correct. Therefore the second base following the anticodon is an A, and not a C as indicated by Koch et al. (1) who used only one figure (fio. 3b in ref. 1) to illustrate the three possible splicing points. It therefore appears that the intron of the maize chloroplast tRNAIle2 gene is located at a position different from that found for the split yeast tRNA gene (44), which is located after the first base following the anticodon. There seems to be no general rule for the position of introns even among maize chloroplast split tRNA genes, as Steinmetz et al. (personal communication) have located an intron after the first base of the anticodon in a maize chloroplast tRNALeu gene. In their article, Koch et al. (1) asked the question whether chloroplast split tRNA genes are expressed. The fact that the tRNAIle we have sequenced is the only tRNAIeIlewhich hybridizes to the DNA of the~~~~~~~~~~~~2spacer region and the fact that it does not hybridize to any other chloroplast DNA restriction fragment (45) show that its gene, which is over 1000 base-pair long, must be transcribed and processed in vivo to yield a mature functional tRNA.

ACKNOWLEDGEMENTS We thank Prof. J. K'ossel for showing us the sequence of the tRNAIle, encoded in the spacer of maize chloroplast DNA and derived from the corresponding gene sequence, prior to publication. We thank Dr. G. Burkard and M. Mubumbila for their help in preparing maize chloroplast tRNAIl2.We are grateful to Dr. R. Giege for providing us with tRNA nucleotidyl transferase.

REFERENCES 1. Koch, W., Edwards, K. and Kossel, H. (1981) Cell 25, 203-213. 2. Chang, S.H., Brum, C.K., Silberklang, M., RajBhandary, U.L., Hecker, L.I. and Barnett, W.E. (1976) Cell 9, 717-725. 3. Guillemaut, P. and Keith, G. (1977) FEBS Lett. 84, 351-356. 4. Canaday, J., Guillemaut, P., Gloeckler, R. and Weil, J.H. (1980) Plant Sci. Lett. 20, 57-62. 5. Osorio-Almeida, M.L., Guillemaut, P., Keith, G., Canaday, J. and Weil,

1657 Nucleic Acids Research

J.H. (1980) Biochem. Biophys. Res. Commun. 92, 102-108. 6. Canaday, J., Guillemaut, P. and Weil, J.H. (1980) Nucl. Acids Res. 8, 999-1008. 7. Calagan, J.L., Pirtle, R.M., Pirtle, I.L., Kashdan, M.A., Vreman, H.J. and Dudock, B.S. (1980) J. Biol. Chem. 255, 9981-9984. 8. McCoy, J.M. and Jones, D.S. (1980) Nucl. Acids Res. 21, 5089-5093. 9. Pirtle, R., Calagan, J., Pirtle, I., Kashdan, M., Vreman, H. and Dudock, B. (1981) Nucl. Acids Res. 9, 183-188. 10. Jones, D.S. (1980) EMBO-FEBS tRNA Workshop, Strasbourg, July 16-21. 11. Kasdan, M.A., Pirtle, R.M., Pirtle, I.L., Calagan, J.L., Vreman, H.J. and Dudock, B.S. (1980) J. Biol. Chem. 255, 8831-8835. 12. McSprouse, H., Kashdan, M., Otis, L. and Dudock, B. (1981) Nucl. Acids Res. 9, 2543-2547. 13. Canaday, J., Guillemaut, P., Gloeckler, R. and Weil, J.H. (1981) Nucl. Acids Res. 9, 47-53. 14. Kenerley, M.E., Morgan, E.A., Post, L., Lindahl, L. and Nomura, M. (1977) J. Bacteriol. 132, 931-949. 15. Kiss, A., Sain, B. and Venetianer, P. (1977) FEBS Lett. 79, 77-79. 16. Ikemura, T. and Nomura, M. (1977) Cell 11, 779-793. 17. Lund, E., Dahlberg, J.E., Lindahl, L., Jaskunas, S.R., Dennis, P.P. and Nomura, M. (1976) Cell 7, 165-177. 18. Morgan, E.A., Ikemura, T., Lindahl, L., Fallon, A.M. and Nomura, M. (1978) Cell 13, 335-344. 19. Young, R.A., Macklis, R. and Steitz, J.A. (1979) J. Biol. Chem. 254, 3264-3271. 20. Sekiya, T. and Nishimura, S. (1979) Nucl. Acids Res. 6, 575-592. 21. Morgan, E.A., Ikemura, T., Post, L.E. and Nomura, M. (1980) in Transfer RNA: Biological Abstracts, Cold Spring Harbor Monograph Series (So11,D., Abelson, J. and Schinmmel, P., eds) vol. 9B, pp. 259-266, Cold Spring Harbor Laboratory, N.Y. 22. Hallick, R.B., Gray, P.W., Chelm, B.K., Rushlow, K.E. and Orozco, E.M. Jr (1978) in Chloroplast Development (Akoyunoglou, G. and Argyroudi- Akoyunoglou, J.H., eds) pp. 619-622, Elsevier/North-Holland, Amsterdam. 23. Hallick, R.B., Rushlow, K.E., Orozco, E.M. Jr, Stiegler, G.L. and Gray, P.W. (1979) ICN-UCLA Symp. Mol. Cell. Biol. 15, 127-141. 24. Orozco, E.M. Jr, Gray, P.W. and Hallick, R.B. (1980) J. Biol. Chem. 255, 10991-10996. 25. Rochaix, J.D. and Malnoe, P. (1978) in Chloroplast Development (Akoyunoglou, G. and Argyroudi-Akoyunoglou, J.H., eds) pp. 581-586, Elsevier/North-Holland, Amsterdam. 26. Malnoe, P. and Rochaix, J.D. (1978) Mol. Gen. Genet. 166, 269-275. 27. Keller, M., Burkard, G., Bohnert, H., Mubumbila, M., Gordon, K., Steinmetz, A., Heiser, D., Crouse, E.J. and Weil, J.H. (1980) Biochem. Biophys. Res. Comnun. 95, 47-54. 28. Bohnert, J.J., Driesel, A.J., Crouse, E.J., Gordon, K., Herrmann, R.G., Steinmetz, A., Mubumbila, M., Keller, M., Burkard, G. and Weil, J.H. (1979) FEBS Lett. 103, 52-56. 29. Orozco, E.M., Rushlow, K.E., Dodd, J.R. and Hallick, R.B. (1980) J. Biol. Chem. 255, 10997-11003. 30. Graf, L., Kossel, H. and Stutz, E. (1980) Nature 286, 908-910. 31. Weil, J.H. (1979) in Nucleic Acids in Plants (Hall, T.C. and Davies, J.W., eds) vol. I, pp. 143-192, CRC Press, Boca Raton, USA. 32. Fradin, A., Gruhl, J. and Feldman, H. (1975) FEBS Lett. 50, 185-189. 33. Driesel, A., Crouse, E.J., Gordon, K., Bohnert, J.H., Herrmann, R.G., Steinmetz, A., Mubumbila, M., Keller, M., Burkard, G. and Weil, J.H. (1979) Gene 6, 285-306. 34. Burkard, G., Steinmetz, A., Keller, M., Mubumbila, M., Crouse, E. and

1658 Nucleic Acids Research

Weil, J.H. in Methods in chloroplast molecular biology (Edeman, M., Hallick, R. and Chua, N., eds) Elsevier/North-Holland (in press). 35. Silberklang, M., Gillum, A.M. and RajBhandary, U.L. (1979) in Methods in Enzymology (Moldave, K. and Grossman, L., eds), vol. LIX, Academic Press, New York, p. 58. 36. Donis-Keller, H., Maxam, A. and Gilbert, W. (1977) Nucl. Acids Res. 4, 2527-2538. 37. Silberklang, M., Gillum, A.M. and RajBhandary, U.L. (1977) Nucl. Acids Res. 4, 4091-4108. 38. Sanger, F. and Coulson, A.R. (1978) FEBS Lett. 87, 107-110. 39. Nishimura, S. (1979) Transfer RNA: Structure, Properties and Recognition (Schimmel, P., So"ll, D. and Abelson, J.N., eds) Cold Spring Harbor monograph series 9A, 551. 40. Stanley, J. and Vassilenko, S. (1978) Nature 274, 87-89. 41. Martin, R.P., Sibler, A.P., Schneller, J.M., Keith, G., Stahl, A.J.C. and Dirheimer, G. (1978) Nucl. Acids Res. 5, 4579-4592. 42. Heckman, J.E., Alzner-deWeerd, B. and RajBhandary, U.L. (1979) Proc. Natl. Acad. Sci. USA 76, 717-721. 43. Gauss, D.H. and Sprinzl, M. (1981) Nucl. Acids Res. 9,r21-r23. 44. Knapp, G., Ogden, R.G., Peebles, C.L. and Abelson, J. (1979 Cell 18, 37-45. 45. Weil, J.H., Guillemaut, P., Burkard, G., Canaday, J., Mubumbila, M., Osorio, M.L., Keller, M., Gloeckler, R., Steirwnetz, A., Keith, G., Heiser, D. and Crouse, E. (1981) Proceedings of the 5th International Photosyn- thesis Congress, "Photosynthesis" (G. Akoyunoglou, ed.), Balaban Interna- tional Science Service, Philadelphia, vol. V, 777-786.

1659