Genetic Approaches to Studying Protein Synthesis: Effects of Mutations at Ψ516 and A535 in Escherichia coli 16S rRNA

A genetic system for the study of ribosomal RNA function and structure was developed. First, the ribosome binding sequence of the chloramphenicol acetyltransferase gene and the message binding sequence of 16S ribosomal RNA were randomly mutated and alternative highly functional sequences were selected and characterized. From this set of mutants, a single clone was chosen and subjected to a second round of mutagenesis to optimize the specificity of the system. In the resulting system, plasmid-encoded ribosomes efficiently and exclusively translate specific mRNA containing the appropriate ribosome binding sequences. This system allows facile isolation and analysis of mutations that would normally be lethal and allows direct selection of rRNA mutants with predetermined levels of ribosome function. The system was used to examine the effects of mutations at the sole pseudouridine (Ψ) in Escherichia coli 16S rRNA which is located at position 516 of the conserved 530 loop. The nucleotide opposite Ψ516 in the hairpin, A535, was also mutated. The data show that a pyrimidine (Ψ or C) is required at position 516, while substitutions at position 535 reduce ribosome function by < 50%. A requirement for base pair formation between Ψ516 and A535 was not indicated.