THE RNA-BINDING SITE OF S8 RIBOSOMAL-PROTEIN OF ESCHERICHIA-COLI - SELEX AND HYDROXYL RADICAL PROBING STUDIES

The RNA binding site of ribosomal protein S8 of Escherichia coli is co nfined to a small region within the stem of a hairpin in 16S rRNA (nt 588-605/633-651), and thus represents a model system for understanding RNA/protein interaction rules. The S8 binding site on 165 rRNA was su spected to contain noncanonical features difficult to prove with class ical genetical or biochemical means. We performed in vitro iterative s election of RNA aptamers that bind S8. For the different aptamers, the interactions with the protein were probed with hydroxyl radicals. Apt amers that were recognized according to the same structural rules as w ild-type RNA, but with variations not found in nature, were identified . These aptamers revealed features in the S8 binding site that had bee n concealed during previous characterizations by the high base conserv ation throughout evolution. Our data demonstrate that the core structu re of the S8 binding site is composed of three interdependent bases (n t 597/641/643), with an essential intervening adenine nucleotide (posi tion 642). The other elements important for the binding site are a bas e pair (598/640) above the three interdependent bases and a bulged bas e at position 595, the identity of which is not important. Possible im plications on the geometry of the S8 binding site are discussed with t he help of a three-dimensional model.