Phosphate sites of RNA-ligands interacting with ribosome at different stages of translation. Thiophosphate method of analysis
Dabrowski, M.; Junemann, R.; Schafer, M.A.; Spahn, C.M.; Nierhaus, K.H.; Alekseeva, E.V.; Dontsova, O.A.; Shpanchenko, O.V.; Bogdanov, A.A.
Biokhimiia 61(11): 1971-1983
1996
ISSN/ISBN: 0320-9725 PMID: 9004858 Document Number: 468384
A novel footprinting method was recently developed which identifies phosphate groups of RNA involved in strong RNA-RNA and RNA-protein interactions. The method is based on iodine-dependent RNA cleavage at phosphothioate groups as long as these groups are not protected from iodine. Our recent studies of mRNA and tRNA regions protected in active ribosomes are summarized; initiation state of ribosomes as well as two elongation states in pre- and post-translocational states were analyzed. Only one phosphate group of mRNA, which was two positions upstream of the decoding codons, was weakly protected in longation complexes, whereas this group and the phosphate groups in the Shine-Dalgarno sequence were protected in the initiation complex. No protection was observed downstream of the decoding codons. On the contrary, numerous phosphate residues of tRNA were protected by the ribosome. The tRNA protection patterns significantly varied between two tRNAs simultaneously bound to the ribosome. The protection pattern of an individual tRNA was not significantly affected by translocation. The data indicate that both tRNA molecules are tightly bound to the ribosome, whereas mRNA is fixed predominantly by two tRNAs via codon-anticodon interaction. A possible translocation mechanism is suggested.