Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA. III. Functional analysis of the 690 loop.

An instant-evolution experiment was performed on the eight nucleotides comprising the loop region of the 690 hairpin in Escherichia coli 16 S ribosomal RNA. Positions 690 to 697 were randomly mutated and 101 unique functional mutants were isolated, sequenced and analyzed for function in vivo. Non-random nucleotide distributions were observed at each of the mutated positions except 693 and 694. Nucleotide identity significantly affected ribosome function at positions 690, 695, 696 and 697. Pyrimidines were absent at position 696 in the instant-evolution pool as were C at position 691 and G at position 697. A highly significant covariation was observed between nucleotides 690 and 697. No functional double mutants at positions 691 and 696 were obtained from the instant-evolution pool. In our NMR structure of the 690 loop, both the G690.U697 and G691.A696 form sheared hydrogen-bonded mismatches. To further examine the functional constraints between these paired nucleotides, one set of site-directed mutations was constructed at positions 690:697 and another set was constructed at positions 691:696. Functional analysis of the site-directed mutants is consistent with our instant-evolution findings and revealed constraints on the placement of specific functional groups observed in the NMR structure. Ten instant-evolution mutants were isolated that are more functional than the wild-type. Hyperactivity in these mutants correlates with a single mutation at position 693.