“…The tentative primary binding site of L24 is the region G298-G301 and C337-G338 (Fig+ 2C, green nucleotides) as inferred from our previous selection studies (Stelzl et al+, 2000a)+ This conclusion is substantiated by the in-gel phosphorothioate probing data+ (1) Phosphorothioate modification of nt A299, G301, and C302 interferes with the L24 binding+ (2) L24 protects the RNA backbone at G298 and A300 from reaction with iodine; the O-to-S substitution at A300 favors binding+ (3) In the 39 part of RNA L4 , an interesting effect is seen with the phosphorothioated G338 that interferes with binding, whereas the minor bound fraction is exposed to iodine cleavage in the L24 complex+ The signals of C334-C337, which cannot be resolved for every base in some experiments, is protected in the L24-rRNA complex+ (4) A G298U transversion that increases the affinity of protein L4 to the rRNA fragment abolishes L24 binding (Stelzl et al+, 2000a)+ Interestingly, only the L24 protections of G298, A300, C334-C337, and A340 and the L4 protection at A320 are similar in the binary and the ternary complexes+ All the other signals change qualitatively when we compare the binary complexes with the ternary complex (Fig+ 2)+ Striking differences between binary and ternary complexes are seen+ (1) Twelve O-to-S substitutions are enriched in the ternary complex, but only two of these (A300 and A320) were enriched in the binary complexes (Fig+ 2D; e+g+, U321, C323, A324)+ Although phosphorothioates are known to increase the affinity for a protein in some cases, the structural meaning is not clear+ It could be that sulfur reduces electrostatic repulsion of the oxygen atoms in the RNA backbone if the molecule is packed tightly, because the R P -sulfur renders the corresponding S P -oxygen less negative (Dertinger et al+, 2000)+ (2) In the loops of helices 19 and 20 some signals from the binary complexes disappear (e+g+, G311, A332), new signals are observed (e+g+, U306, A330) and some signals change (e+g+, A309, G313)+ (3) Striking changes are even observed in the protein interaction sites: At the L24 binding site the interferences are either lost (e+g+, G301, C302; Fig+ 2D) or replaced by protection signals (G297, A299, G338)+ The phosphorothioate 59 to U321 that abolishes L4 binding to the RNA becomes protected and, most astonishingly, is now enriched in the ternary complex+ (4) Apparently conflicting results are found in the binary complexes for two positions in the L4 binding region: An A320 modification that is enriched in the rRNA L4 -L4 binary complex but interferes with L24-rRNA L4 complex formation is also enriched in the ternary complex+ The opposite case is seen with thioated A322, which gives an interference signal in the binary complex with L4 and is enriched in that with L24+ In the ternary complex the interference effect is seen; as with L4 alone, thioated A322 also impedes ternary complex formation+ These observations demonstrate an RNA conformational change when going from one of the binary complexes to the ternary complex+…”