Contact Patterns of RNA Ligands with the Ribosome in Defined Functional States as Determined by Protection against Cleavage at Phosphorothioated Residues: a Review

M. Dabrowski,¹ R. Junemann,¹ M. A. Schafer,¹ C. M. T. Spahn,¹ K. H. Nierhaus,^1,2 E. V. Alexeeva,^1,3 O. A. Dontsova,³ O. V. Shpanchenko,^1,3 and A. A. Bogdanov³

¹Max-Planck-Institut fur Molekulare Genetik, AG Ribosomen, Ihnestr. 73, D-14195 Berlin, Germany; fax: (30) 84-13-80; E-mail: nierhausKH@mpimg-berlin-dahlem.mpg.de

²To whom correspondence should be addressed.

³School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (7-095) 939-31-81; E-mail: dontsova@ribosome.genebee.msu.su

Submitted July 31, 1996.

A few years ago a new footprinting method was introduced which identifies contacts at the phosphate groups of RNAs. The method exploits the potential of iodine (I₂) to cleave at phosphorothioated positions of RNAs as long as no other components prevent access of the iodine molecule to the corresponding phosphorothioate group. Here we give a survey of recent experiments where the contact patterns of mRNA and tRNAs within active ribosomes have been explored. The initiation state of ribosomes as well as the two elongation states, viz. the pre- and the post-translocational states, have been analyzed. Only weak protections were found for the mRNA. In the elongation complexes the phosphate group two positions upstream of the decoding codons was protected, whereas in the initiation complex phosphate groups in the Shine-Dalgarno sequence were also protected. No protection could be seen downstream of the decoding codons. In contrast, numerous phosphate positions were protected in tRNAs bound to the ribosome. The tRNA protection patterns were highly differentiated and were strikingly different for the two tRNAs simultaneously bound to the ribosome. The protection pattern of an individual tRNA remained almost constant upon translocation. The results suggest that the ribosome might hold the tRNAs by a whole set of direct contacts, whereas the mRNA is mainly fixed at the two decoding codons and by the tRNAs via codon--anticodon interaction. A hypothesis about a possible mechanism of the translocation process is discussed.

KEY WORDS: ribosome, mRNA, tRNA, thiophosphates, footprinting, elongation.

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