title: Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 11. Enzymatic joining to form the total DNA duplex.
abstract: The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D005796:Genes; D007700:Kinetics; D009690:Nucleic Acid Conformation; D010744:Phosphoric Monoester Hydrolases; 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
chemical_list: D004269:DNA, Bacterial; D014443:Tyrosine; D012343:RNA, Transfer; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D010744:Phosphoric Monoester Hydrolases; D011118:Polynucleotide Ligases