Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 7. Enzymatic joining of the chemically synthesized segments to form a DNA duplex corresponding to the nucleotide sequence 1-26.
Duplex [I], which represents the nucleotide sequence 1-26 of the double-stranded DNA corresponding to the precursor for a tyrosine suppressor tRNA, has been synthesized by the enzymatic joining of five chemically synthesized deoxyribooligonucleotide segments. The synthesis was accomplished in two different ways. In a one-step synthesis, all of the five segments were used together: segments 2, 3, and 5 carried 5'-33P-labeled phosphate groups while segment 4 carried a 32P-phosphate group. An alternative, two-step method involved the joining of 5'-32P-phosphorylated segment 2 to segment 4 (carrying 5'-OH group or 5'-32P- or 33P-labeled phosphate group) in the presence of segment 3 followed by the joining of [5-32P]segment 5 in a second step. The duplex [I]' (segments 2 to 5) thus obtained was phosphorlated at the 5'-ends with polynucleotide kinase and then joined to segment 1 to give duplex [I] quantitatively. The preparative methods described have the desired flexibility for performing the subsequent operations necessary for the total synthesis of the structural gene for the tyrosine suppressor tRNA precursor.
D001483:Base Sequence; D003090:Coliphages / Q000201:enzymology; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D005796:Genes; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D011089:Polydeoxyribonucleotides / Q000096:biosynthesis*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D011118:Polynucleotide Ligases / Q000378:metabolism*; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000096:biosynthesis; D014158:Transcription, Genetic; D014443:Tyrosine / Q000096:biosynthesis
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D011089:Polydeoxyribonucleotides; D014443:Tyrosine; D012343:RNA, Transfer; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D011118:Polynucleotide Ligases