Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro.
A recombinant plasmid was constructed with six synthetic DNA oligomers such that the DNA sequence corresponding to yeast tRNA(Phe) is flanked by a T7 promoter and a BstNI restriction site. Runoff transcription of the BstNI-digested plasmid with T7 RNA polymerase gives an unmodified tRNA of the expected sequence having correct 5' and 3' termini. This tRNA(Phe) transcript can be specifically aminoacylated by yeast phenylalanyl-tRNA synthetase and has a Km only 4-fold higher than that of the native yeast tRNA(Phe). The Km is independent of Mg2+ concentration, whereas the Vmax is very dependent on Mg2+ concentration. Comparison of the melting profiles of the native and the unmodified tRNA(Phe) at different Mg2+ concentrations suggests that the unmodified tRNA(Phe) has a less stable tertiary structure. Using one additional DNA oligomer, a mutant plasmid was constructed having a guanosine to thymidine change at position 20 in the tRNA gene. A decrease in Vmax/Km by a factor of 14 for aminoacylation of the mutant tRNA(Phe) transcript is observed.
Sampson
J R
JR
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
Uhlenbeck
O C
OC
D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics; D004274:DNA, Recombinant; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D010649:Phenylalanine / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics* / Q000378:metabolism; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics*; D012360:RNA, Transfer, Phe / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004271:DNA, Fungal; D004274:DNA, Recombinant; D012331:RNA, Fungal; D012345:RNA, Transfer, Amino Acid-Specific; D012360:RNA, Transfer, Phe; D010649:Phenylalanine; D010652:Phenylalanine-tRNA Ligase