COOPERATIVE ASSEMBLY OF PROTEINS IN THE RIBOSOMAL GTPASE CENTER DEMONSTRATED BY THEIR INTERACTIONS WITH MUTANT 23S RIBOSOMAL-RNAS

The ribosomal protein L11 binds to the region of 23S rRNA associated w ith the GTPase-dependent steps of protein synthesis, Nucleotides 1054- 1107 within this region of the Escherichia coil 23S rRNA gene were mut agenized with bisulphite, Twenty point mutations (G --> A and C-->T tr ansitions) and numerous multiple mutations were generated, Expression of mutant 23S rRNAs in vivo shows that all the mutations detectably al ter the phenotype, with effects ranging from a slight growth rate redu ction to lack of viability, Temperature sensitivity is conferred by 10 71G --> A and 1092C --> U substitutions, These effects are relieved by point mutations at other sites, indicating functional interconnection s within the higher order structure of this 23S rRNA region, Several m utations prevent direct binding of r-protein L11 to 23S rRNA in vitro, These mutations are mainly in a short irregular stem (1087-1102) and within a hairpin loop (1068-1072), where the protein probably makes nu cleotide contacts, Some of these mutations also interfere with binding of the r-protein complex L10.(L12)(4) to an adjacent site on the rRNA , When added together to rRNA, proteins L10.(L12)(4) and L11 bind coop eratively to overcome the effects of mutations at 1091 and 1099, The p roteins also stimulate each others binding to rRNA mutated at 1087 or 1092, although in these cases binding remains clearly substoichiometri c, Surprisingly, none of the mutations prevents incorporation of L11 i nto ribosomes in vivo, indicating that other, as yet unidentified, fac tors are involved in the cooperative assembly process.