MUTATIONS AT 2 INVARIANT NUCLEOTIDES IN THE 3'-MINOR DOMAIN OF ESCHERICHIA-COLI 16S RIBOSOMAL-RNA AFFECTING TRANSLATIONAL INITIATION AND INITIATION-FACTOR-3 FUNCTION

We have investigated the highly conserved GAUCA sequence of small subu nit ribosomal RNA. Within this region, the invariant nucleotides G1530 and A1531 of Escherichia coli 16 S rRNA were mutagenized to A1530/G15 31, These base changes caused a lethal phenotype when expressed from a high copy number plasmid, In low copy number plasmids, the mutant rib osomes had limited effects when expressed in vivo but caused significa nt deficiencies in translation in vitro, affecting enzymatic tRNA bind ing, non-enzymatic tRNA binding, subunit association, and initiation f actor 3 (IF3) binding, Mutant 30 S ribosomal subunits showed a 10-fold decrease in affinity for IF3 as compared to wild-type subunits but sh owed an increased affinity for IF3 when in 70 S ribosomes, Additionall y, IF3 did not promote dissociation of 70 S ribosomes, which had mutat ed subunits as monitored by light-scattering experiments, However, ext ension inhibition experiments (toeprinting) showed that IF3 retained i ts ability to discriminate between initiator and elongator tRNAs on mu tated subunits, The results indicate that the two functions of IF3, tR NA discrimination and subunit dissociation, are separable and that the invariant nucleotides are important for correct subunit function duri ng initiation.