Minimal 16S rRNA binding site and role of conserved nucleotides in Escherichia coli ribosomal protein S8 recognition

Escherichia coli ribosomal protein S8 was previously shown to bind a 16S rRNA fragment (nucleotides 584–756) with the same affinity as the complete 16S rRNA, and to shield an irregular helical region (region C) [Mougel, M., Eyermann, F., Westhof, E., Romby, P., Expert-Bezancon, Ebel, J. P., Ehresmann, B. & Ehresmann, C. (1987). J. Mol. Biol. 198, 91–107]. Region C was postulated to display characteristic features: three bulged adenines (A595, A640 and A642), a non-canonical U598–U641 pair surrounded by two G · C pairs. In order to delineate the minimal RNA binding site, deletions were introduced by site-directed mutagenesis and short RNA fragments were synthesized. Their ability to bind S8 was assayed by filter binding. Our results show that the RNA binding site can be restricted to a short helical stem (588–605/633–651) containing region C. The second part of the work focused on region C and on the role of conserved nucleotides as potential determinants of S8 recognition. Single and double mutations were introduced by site-directed mutagenesis in fragment 584–756, and their effect on S8 binding was measured. It was found that the three bulged positions are essential and that adenines are required at positions 640 and 642. U598 is also crucial and the highly conserved G597 · C643 pair cannot be inverted. These conserved nucleotides are either directly involved in the recognition process as direct contacts or required to maintain a specific conformation. The strong evolutionary pressure and the small number of positive mutants stress the high stringency of the recognition process.