Effect of buffer conditions on the position of tRNA on the 70 S ribosome as visualized by cryoelectron microscopy

The effect of buffer conditions on the binding position of tRNA on the Esch erichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy. Either deacylated tRNA(f)(met) or fMet-tRNA(f )(Met) were bound to the 70 S ribosomes, which were programmed with a 46-nu cleotide mRNA having AUG codon in the middle, under two different buffer co nditions (conventional buffer: containing Tris and higher Mg2+ concentratio n [10-15 mM]; and polyamine buffer: containing Hepes, lower Mg2+ concentrat ion [6 mM], and polyamines). Difference maps, obtained by subtracting 3D ma ps of naked control ribosome in the corresponding buffer from the 3D maps o f tRNA ribosome complexes, reveal the distinct locations of tRNA on the rib osome. The position of deacylated tRNA(f)(Met) depends on the buffer condit ion used, whereas that of fMet-tRNA(f)(Met) remains the same in both buffer conditions. The acylated tRNA binds in the classical P site, whereas deacy lated tRNA binds mostly in an intermediate P/E position under the conventio nal buffer condition and mostly in the position corresponding to the classi cal P site, i.e. in the P/P state, under the polyamine buffer conditions.