Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 10. Enzymatic joining of chemically synthesized segments to form the DNA duplex corresponding to the nucleotide sequence 86-126.
The polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxypolynucleotides (segments 19 to 26), comprising the nucleotide sequence 86-126 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been investigated. Joining was studied using various combinations of 3, 4, or larger number of segments at a time. The extent of joining was in general low (0 to 40%) for the three-component as well as for the four-component systems. Joining of the five- and six- component systems was more satisfactory with yields from 25 to about 60%. The three duplexes [IVa] to [IVc]were prepared in single step reactions in yields of about 50% and were characterized. Duplex [IVd] could not be prepared in a single step reaction because of the failure of 5'-phosphorylated segment 26 to join to the rest of the duplex. Using a carefully annealed mixture of segments 24, 25, and phosphorylated segment 26, the joining of the latter to segment 24 could be realized in about 25% yield, much activated intermediate being concurrently present.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D005796:Genes; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010744:Phosphoric Monoester Hydrolases; 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; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D010744:Phosphoric Monoester Hydrolases; D011118:Polynucleotide Ligases