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Characterization of the fluorescent bimane derivative of E. coli initiator transfer RNA (tRNAfMet)

1985; Elsevier BV; Volume: 127; Issue: 1 Linguagem: Inglês

10.1016/s0006-291x(85)80124-8

1090-2104

C. Pande, Arnold Wishnia,

RNA modifications and cancer

The invariant modified base 4-thiouridine of the E. Coli initiator tRNA was chemically modified using a sulfhydryl specific fluorogenic probe, monobromobimane. The modified tRNAfMet is virtually indistinguishable biochemically from the native form in the aminoacylation and formylation reactions, and in its binding behavior to the ribosomal P site. Fluorescence quenching by I- increases 40% when the modified tRNA is charged with formylmethionine, even at this relatively well-shielded position in the tRNA elbow. Most important, the fluorescence polarization increases by a factor of 2, to almost the irrotational value, when fMet-tRNAfMet binds to the ribosomal P site, providing a useful tool for studying fMet-tRNAfMet-ribosome interaction equilibria and kinetics.