Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 8. Enzymatic joining of the chemically synthesized segments to form DNA duplexes corresponding to nucleotide sequences 23-60 and 23-66.
Polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxyribopolynucleotide segments (Fig. 1) comprising the nucleotide sequence 23-66 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been systematically investigated. Joining was studied using all possible combinations of 3, 4, and 5 and larger numbers of segments at a time. The extent of joining varied widely (0 to about 90%) in three component systems. The "self-structure" of some of the components evidently inhibited the joining. Addition of a fourth segment in general enhanced the extent of joining and optimal yields were obtained in systems containing six or more segments. A comparison of the T4-induced ligase and the E. coli polynucleotide ligase for joining of the chemically synthesized segments showed the E. coli enzyme to be inferior to the T4-induced ligase. Satisfactory syntheses of the duplexes [IIa] and [IIb] comprising, respectively, eight and seven segments were achieved in single steps. Of the two terminal segments carrying 5'-OH groups in the duplexes, only one (segment 7) was used in the prephosphorylated form. The duplexes were isolated pure and characterized by enzymatic degradations and by electrophoresis.
D001483:Base Sequence; D003090:Coliphages / Q000201:enzymology; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D005796:Genes; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis; D011118:Polynucleotide Ligases / Q000378:metabolism*; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014443:Tyrosine / Q000096:biosynthesis
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D011089:Polydeoxyribonucleotides; D012329:RNA, Bacterial; D014443:Tyrosine; D011118:Polynucleotide Ligases