Abstract
The effect of buffer conditions on the binding position of tRNA on the Escherichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy. Either deacylated tRNAfMet or fMet-tRNAfMet were bound to the 70 S ribosomes, which were programmed with a 46-nucleotide mRNA having AUG codon in the middle, under two different buffer conditions (conventional buffer: containing Tris and higher Mg2+ concentration [10-15 mM]; and polyamine buffer: containing Hepes, lower Mg2+ concentration [6 mM], and polyamines). Difference maps, obtained by subtracting 3D maps of naked control ribosome in the corresponding buffer from the 3D maps of tRNA.ribosome complexes, reveal the distinct locations of tRNA on the ribosome. The position of deacylated tRNAfMet depends on the buffer condition used, whereas that of fMet-tRNAfMet remains the same in both buffer conditions. The acylated tRNA binds in the classical P site, whereas deacylated tRNA binds mostly in an intermediate P/E position under the conventional buffer condition and mostly in the position corresponding to the classical P site, i. e. in the P/P state, under the polyamine buffer conditions.
Highlights
The ribosome coordinates and facilitates the biosynthesis of the polypeptide chain by the addition of new amino acids, which are brought to the ribosome by transfer RNAs, according to genetic instructions stored on the messenger RNA. tRNA is known to occupy successive positions in the intersubunit space of the ribosome in the course of the elongation cycle
In the polyamine buffer system (Fig. 2d), on the other hand, the reverse situation is observed; a strong mass of density is found in a position superimposing with the fMet-tRNAfMet and a very weak mass of density is found on the L1 side
The only feature that the two tRNAs, in the P/P and P/E positions, have in common is that they share the region where the anticodon of the P-site tRNA interacts with the messenger RNA (mRNA) codon, whereas the rest of the tRNA molecule is located in different ways (Fig. 5)
Summary
The ribosome coordinates and facilitates the biosynthesis of the polypeptide chain by the addition of new amino acids, which are brought to the ribosome by transfer RNAs (tRNAs), according to genetic instructions stored on the messenger RNA (mRNA). tRNA is known to occupy successive positions in the intersubunit space (see Ref. 1) of the ribosome in the course of the elongation cycle. Of the various tRNA binding positions on the ribosome, the position of P-site tRNA has been unambiguously defined in a 3D map of the fMet-tRNAfMet1⁄7ribosome complex at 15 Å resolution [8] Because this position differs from the position of P-site tRNA inferred from an earlier experiment in our laboratory [4], a clarification of the cause of the discrepancy was needed. As in these two studies [4, 8], buffer conditions (conventional versus polyamine), state of the tRNA (deacylated versus acylated), as well as the mRNA (poly(U) versus MF-mRNA, an mRNA with a defined codon) were different, additional experiments were required to answer this question conclusively. This article presents the cryoelectron microscopy results of experiments in which two buffer conditions (conventional and polyamine) and two states of tRNAs (aminoacylated versus deacylated) were used to investigate the properties of tRNA at the ribosomal P site
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