Construction of a composite tRNA gene by anticodon loop transplant.
By using sites for the restriction nuclease Hpa II, the information for the anticodon stem and loop of an altered Su+2 amber suppressor tRNA (a mutant of tRNAGln) has been transplanted to a specially prepared tRNATrp gene, which lacks it homologous anticodon stem and loop sequence. The resulting tRNA gene was cloned under lac operator-promoter control. The result is a functional, hybrid, amber-suppressor tRNA that can exhibit a moderately high efficiency in translation. It appears less efficient, however, than Su+7 tRNA, the amber suppressor that results from a direct anticodon mutation in tRNATrp. As judged by its suppressor spectrum, which is almost identical to the spectra of Su+2, and Su+7, the recomposed tRNA inserts glutamine at amber sites. This experiment is the prototype of a series of construction that examine the role of the nucleotides in the anticodon region.
D000926:Anticodon; D010582:Bacteriophage lambda; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008025:Ligases; D008722:Methods; D010957:Plasmids; D012343:RNA, Transfer / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic; D013489:Suppression, Genetic; D014170:Transformation, Genetic
D000926:Anticodon; D012343:RNA, Transfer; D004262:DNA Restriction Enzymes; D008025:Ligases