CXIII. Total synthesis of the structural gene for an alanine transfer RNA from yeast. Enzymic joining of the chemically synthesized segments to form the DNA duplex corresponding to nucleotide sequence 17 to 50

Abstract

The DNA duplex (designated [B] and shown in Fig. 1), corresponding to the middle part (nucleotide sequence 17 to 50) of the major yeast alanine transfer RNA has been synthesized. The duplex contained short single-stranded regions at both ends for subsequent joining to the [A](paper CXII) and [C](paper CXIV) parts of the total DNA duplex. The T4 ligase was shown to bring about the joining of d-(5′- 32P)-C-T-A-A-G (segment 9) to the icosanucleotide (segment 6) in the presence of the complementary segment 8 (Fig. 1); similarly, d-(5′- 32P)-A-G-A-G-T-C-T (segment 7) could be joined efficiently to segment 8 in the presence of segment 6. The synthesis of [B] containing the five components (segments 5 to 9) was carried out as follows: the joining of segment 9 to segment 6 and that of segment 7 to segment 8 was carried out in one step at 15 °C. Without isolation of the resulting duplex, 5′- 32P-labeled segment 5 was added and the incubation continued at 25 °C. The joining of segment 7 was complete at this temperature. As required for joining [B] to the two terminal parts, [A] and [C] of the DNA, [B] as obtained above was enzymically phosphorylated at the two 5′-terminal deoxyguanosine units using γ-labeled ATP and polynucleotide kinase and the product was isolated by gel-filtration.