Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 12. Synthesis of a DNA duplex corresponding to a sequence of 23 nucleotide units adjoining the C-C-A end

Abstract

In continuing the work on the total synthesis of the gene for an Escherichia coli tyrosine suppressor tRNA (accompanying papers) and as a part of a study of the mechanism of transcription of this gene, a 23-nucleotide unit-long DNA corresponding to the previously determined (Loewen, P., Sekiya, T., and Khorana, H. G. (1974) J. Biol. Chem. 249, 217) sequence has been synthesized. The synthesis was carried out by dividing the total duplex into the following five deoxyribooligonucleotide segments, all of which were chemically synthesized: (a) the undecanucleotide, d(A-G-T-G-A-T-G-G-T-G-G); (b)the undecanucleotide, d(T-C-A-C-T-T-T-C-A-A-A); (c) the undecanucleotide, d(G-G-A-C-T-T-T-T-G-A-A); (d) the dodecanucleotide, d(A-G-T-C-C-C-T-G-A-A-C-T); and (e) the heptanucleotide, d(A-G-T-T-C-A-G). All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling the 5'-ends with a 32P-phosphate group. Synthesis of the double-stranded DNA duplex was completed by joining 5'-phosphorylated segments 1, 3, and 4 in the presence of segments 2 and 5 using T4-polynucleotide ligase. The DNA duplex was characterized.