Synthesis of 5' fragments of formylmethionine transfer ribonucleic acid and their reconstitution with a natural three-quarter molecule.
An eicosanucleotide C--G--C--G--G--G--G--U--G--G--A--G--C--A--G--C--C--U--G--Gp corresponding to the bases 1--20 of the nascent sequence for the Escherichia coli tRNAfMet has been synthesized by the joining of the chemically synthesized oligonucleotides C--G--C--G, G--G--G--U--G--G and A--G--C--A--G--C--C--U--G--Gp using RNA ligase from T4-infected E. coli. The hexanucleotide and decanucleotide were phosphorylated with polynucleotide kinase and [gamma-32P]ATP prior to the joining reactions. The decanucleotide and eicosanucleotide were reconstituted respectively with the 3'-three-quarter molecule obtained by limited digestion with RNase T1 of the natural tRNAfMet from E. coli and the activity of the complex as a methionine acceptor was tested using purified methionyl-tRNA synthetase from E. coli. The amino acid acceptor activity of the reconstituted molecules was 11% and 84% with respect to that of the intact tRNAfMet.
D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism*; D009239:N-Formylmethionine; D009841:Oligonucleotides / Q000138:chemical synthesis*; D009843:Oligoribonucleotides / Q000138:chemical synthesis*; D011130:RNA Ligase (ATP); D012329:RNA, Bacterial; D012343:RNA, Transfer; D013604:T-Phages / Q000201:enzymology
D009841:Oligonucleotides; D009843:Oligoribonucleotides; D012329:RNA, Bacterial; D009239:N-Formylmethionine; D012343:RNA, Transfer; D011130:RNA Ligase (ATP)