In vivo determination of RNA structure-function relationships: analysis of the 790 loop in ribosomal RNA

The 790 loop is a conserved hairpin located between positions 786 and 796 of Escherichia coli 16 S rRNA that is required for ribosome function. Using a novel genetic approach, all positions in the loop were simultaneously mutated and functional mutant sequences were selected in vivo. This "instant evolution" experiment revealed that approximately 190 of the 262,144 possible mutant sequences were functional. Analysis of functional mutant sequences allowed discrimination between nucleotides directly involved in protein synthesis and those involved primarily in loop structure. Among the functional mutant sequences, positions 789 and 791 were invariant and extensive covariation was observed among the nucleotides at the base of the loop at positions 787, 788, 794 and 795. NMR and thermodynamic analyses of model 790 hairpins in vitro revealed weak pairing interactions between positions 787 and 795 and between positions 788 and 794 consistent with the in vivo mutational analysis. Functional analysis of site-directed mutants containing all possible nucleotide combinations at positions 787 and 795 in vivo showed that stable base-pairs at these positions prevent subunit association.