The Nucleotide Sequence Adjoining the CCA End of an Escherichia coli Tyrosine Transfer Ribonucleic Acid Gene

The sequence of 23 nucleotides beyond the C-C-A end of the Escherichia coli tyrosine tRNA gene has been determined. The previously deduced (Loewen, P. C., and Khorana, H. G. (1973) J. Biol. Chem. 248, 3489) sequence of the first 12 nucleotides (T-C-A-A-C-T-T-T-C-A-A-A) has been revised to T-C-A-C-T-T-T-C-A-A-A-A. The sequence of the next 11 nucleotides is G-T-C-C-C-T-G-A-A-C-T. The general approach used in the sequence work was the DNA polymerase-I-catalyzed elongation of suitable deoxyribopolynucleotides which served as primers when hybridized to the r-strand of φ80psuiii DNA. Five methods were used in the sequential analysis of the elongated primers: (a) the use of a restricted number of deoxyribonucleoside triphosphates; (b) timed incorporation of all of the four deoxyribonucleoside triphosphates and isolation of the pure radioactive products by polyacrylamide gel electrophoresis; (c) isolation of the products from timed incorporation of the dNTPs in which CTP replaced dCTP and Mn2+ replaced Mg2+; (d) separation of the fragments formed after alkaline hydrolysis by two-dimensional electrophoresis; (e) derivation of the nucleotide sequence after partial stepwise degradation by spleen and venom phosphodiesterases by two-dimensional finger-printing methods of Sanger and co-workers (Sanger, F., Donelson, J. E., Coulson, A. R., Kossel, H., and Fischer, D. (1973) Proc. Nat. Acad. Sci. U. S. A. 70, 1209).