Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

The many interactions of tRNA with the ribosome are fundamental to protein synthesis. During the peptidyl transferase reaction, the acceptor ends of t he aminoacyl and peptidyl tRNAs must be in close proximity to allow peptide bond formation, and their respective anticodons must base pair simultaneou sly with adjacent trinucleotide codons on the mRNA, The two tRNAs in this s tate can be arranged in two nonequivalent general configurations called the R and S orientations, many versions of which have been proposed for the ge ometry of tRNAs in the ribosome. Here, we report the combined use of comput ational analysis and tethered hydroxyl-radical probing to constrain their a rrangement. We used Fe(II) tethered to the 5' end of anticodon stem-loop an alogs (ASLs) of tRNA and to the 5' end of deacylated tRNA(Phe) to generate hydroxyl radicals that probe proximal positions in the backbone of adjacent tRNAs in the 70S ribosome. We inferred probe-target distances from the res ulting RNA strand cleavage intensities and used these to calculate the mutu al arrangement of A-site and P-site tRNAs in the ribosome, using three diff erent structure estimation algorithms, The two tRNAs are constrained to the S configuration with an angle of about 45 degrees between the respective p lanes of the molecules. The terminal phosphates of 3'CCA are separated by 2 3 Angstrom when using the tRNA crystal conformations, and the anticodon arm s of the two tRNAs are sufficiently close to interact with adjacent codons in mRNA.