In vitro evolution used to define a protein recognition site within a large RNA domain Original Research Article

A minimum of 460 nucleotides of 16S ribosomal RNA are needed to fold the target site for E. coli ribosomal protein S4, although a much smaller region within this large domain is protected from chemical reagents by the protein. Starting with a 531-nucleotide rRNA fragment, cycles of mutagenesis, selection with S4, and amplification (‘in vitro evolution’) were used to obtain a pool of 30 RNA sequences selected for S4 recognition but ∼30% different from wild type. Numerous compensatory base pair changes have largely preserved the same secondary structure among these RNAs as found in wild-type sequences. A 20-base deletion and a single nucleotide insertion are among several unusual features found in most of the selected sequences and also prevalent among other prokaryotic rRNAs. Most of the compensatory base changes and selected features are located outside of the region protected by S4 from chemical reagents. It was unexpected that S4 would select for RNA structures throughout such a large domain; the selected features are probably contributing indirectly to S4 recognition by promoting correct tertiary folding of the region actually contacted by S4. The role of S4 may be to stabilize this domain (nearly one-third of the 16S rRNA) in its proper conformation for ribosome function.