A synthetic alanyl-initiator tRNA with initiator tRNA properties as determined by fluorescence measurements: comparison to a synthetic alanyl-elongator tRNA.
Two synthetic tRNAs have been generated that can be enzymatically aminoacylated with alanine and have AAA anticodons to recognize a poly(U) template. One of the tRNAs (tRNA(eAla/AAA)) is nearly identical to Escherichia coli elongator tRNA(Ala). The other has a sequence similar to Escherichia coli initiator tRNA(Met) (tRNA(iAla/AAA)). Although both tRNAs can be used in poly(U)-directed nonenzymatic initiation at 15 mM Mg2+, only the elongator tRNA can serve for peptide elongation and polyalanine synthesis. Only the initiator tRNA can be bound to 30S ribosomal subunits or 70S ribosomes in the presence of initiation factor 2 (IF-2) and low Mg2+ suggesting that it can function in enzymatic peptide initiation. A derivative of coumarin was covalently attached to the alpha amino group of alanine of these two Ala-tRNA species. The fluorescence spectra, quantum yield and anisotropy for the two Ala-tRNA derivatives are different when they are bound to 70S ribosomes (nonenzymatically in the presence of 15 mM Mg2+) indicating that the local environment of the probe is different. Also, the effect of erythromycin on their fluorescence is quite different, suggesting that the probes and presumably the alanine moiety to which they are covalently linked are in different positions on the ribosomes.
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Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
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D001483:Base Sequence; D003374:Coumarins / Q000378:metabolism; D004917:Erythromycin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010441:Peptide Chain Elongation, Translational; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D039881:Prokaryotic Initiation Factor-2; D014176:Protein Biosynthesis; D011691:Puromycin / Q000378:metabolism; D012344:RNA, Transfer, Ala / Q000138:chemical synthesis* / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000138:chemical synthesis / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013033:Sparsomycin / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D003374:Coumarins; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D012344:RNA, Transfer, Ala; D012358:RNA, Transfer, Met; D011072:Poly U; D011691:Puromycin; D004917:Erythromycin; D013033:Sparsomycin