ROLE OF CONSERVED NUCLEOTIDES IN BUILDING THE 16S RIBOSOMAL-RNA BINDING-SITE OF ESCHERICHIA-COLI RIBOSOMAL-PROTEIN S8

Ribosomal protein S8 specifically recognizes a helical and irregular r egion of 16S rRNA that is highly evolutionary constrained. Despite its restricted size, the precise conformation of this region remains a qu estion of debate. Here, we used chemical probing to analyze the struct ural consequences of mutations in this RNA region. These data, combine d with computer modelling and previously published data on protein bin ding were used to investigate the conformation of the RNA binding site . The experimental data confirm the model in which adenines A595, A640 and A642 bulge out in the deep groove. In addition to the already pro posed non canonical U598 - U641 interaction, the structure is stabiliz ed by stacking interactions (between A595 and A640) and an array of hy drogen bonds involving bases and the sugar phosphate backbone. Mutatio ns that alter the ability to form these interdependent interactions re sult in a local destabilization or reorganization. The specificity of recognition by protein S8 is provided by the irregular and distorted b ackbone and the two bulged adenines 640 and 642 in the deep groove. Th e third adenine (A595) is not a direct recognition site but must adopt a bulged position. The U598 - U641 pair should not be directly in con tact with the protein.