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 confined 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 16S rRNA was suspected to contain noncanonical features difficult to prove with classical genetical or biochemical means. We performed in vitro iterative selection of RNA aptamers that bind S8. For the different aptamers, the interactions with the protein were probed with hydroxyl radicals. Aptamers that were recognized according to the same structural rules as wild-type RNA, but with variations not found in nature, were identified. These aptamers revealed features in the S8 binding site that had been concealed during previous characterizations by the high base conservation throughout evolution. Our data demonstrate that the core structure of the S8 binding site is composed of three interdependent bases (nt 597/641/643), with an essential intervening adenine nucleotide (position 642). The other elements important for the binding site are a base pair (598/640) above the three interdependent bases and a bulged base at position 595, the identity of which is not important. Possible implications on the geometry of the S8 binding site are discussed with the help of a three-dimensional model.
Moine
H
H
UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France. moine@ibmc.u-strasbg.fr.
Cachia
C
C
Westhof
E
E
Ehresmann
B
B
Ehresmann
C
C
D001345:Autoradiography; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004926:Escherichia coli; D005821:Genetic Techniques; D017665:Hydroxyl Radical / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; C030254:ribosomal protein S8; D017665:Hydroxyl Radical