Role of conserved nucleotides in building the 16 S rRNA binding site for ribosomal protein S15

Ribosomal protein S15 recognizes a highly conserved target on 16 S rRNA, wh ich consists of two distinct binding regions. Here, we used extensive site- directed mutagenesis on a Escherichia coli 16 S rRNA fragment containing th e S15 binding site, to investigate the role of conserved nucleotides in pro tein recognition and to evaluate the relative contribution of the two sites . The effect of mutations on S15 recognition was studied by measuring the r elative binding affinity, RNA probing and footprinting. The crystallographi c structure of the Thermus thermophilus complex allowed molecular modelling of the E. coli complex and facilitated interpretation of biochemical data. Binding is essentially driven by site 1, which includes a three-way juncti on constrained by a conserved base triple and cross-strand stacking. Recogn ition is based mainly on shape complementarity, and the role of conserved n ucleotides is to maintain a unique backbone geometry. The wild-type base tr iple is absolutely required for protein interaction, while changes in the c onserved surrounding nucleotides are partially tolerated. Site 2, which pro vides functional groups in a conserved G-U/G-C motif, contributes only mode stly to the stability of the interaction. Binding to this motif is dependen t on binding at site 1 and is allowed only if the two sites are in the corr ect relative orientation. Non-conserved bulged nucleotides as well as a con served purine interior loop, although not directly involved in recognition, are used to provide an appropriate flexibility between the two sites. In a ddition, correct binding at the two sites triggers conformational adjustmen ts in the purine interior loop and in a distal region, which are known to b e involved for subsequent binding of proteins S6 and S18. Thus, the role of site 1 is to anchor S15 to the rRNA, while binding at site 2 is aimed to i nduce a cascade of events required for subunit assembly. (C) 2001 Academic Press.