The effects of antibiotics and mutations on the decoding accuracy of translation and the revelation of A-site and P-site reciprocation

Initiation of translation is a unique from the elongation phase in that it occurs in the 30s with three initiation factors IF1, IF2, and IF3. The decoding process of translation has been studied largely from the perspective of elongation. Here we describe a unified mechanism for IF3, identify antibiotics that are able to specifically decrease decoding accuracy during initiation, and provide evidence for our reciprocation model for A and P site interdependence. Antibiotics and mutations that have a Ram effect on the A-site have been previously identified and used as tools for studying translation and decoding accuracy. Here we study mutations and identify antibiotics that alter decoding accuracy during initiation and then provide evidence for a direct interdependence of A and P-site accuracy.; Lac-based reporters containing no leader or near-cognate start codons were used to measure P-site decoding accuracy and those having an early stop codon were used to measure A-site decoding accuracy. F-Met-tRNAfMet selection was measured using in vitro---Toeprinting assays. In order to test the reciprocation model growth was followed combining agents that decrease A and P-site decoding accuracy.; We show that N-terminal mutations in IF3 are able to disturb accuracy in three different ways during initiation, tRNA selection, leaderless expression, and start codon recognition and suggest a unified model for IF3 function whereby IF3 acts to destabilize the initiation complex, thus selecting against incorrect initiation complexes. We identify the antibiotics kasugamycin, spectinomycin, pactamycin, and tetracycline as P-site RAM antibiotics. In support of the reciprocation model, we show that ribosomal mutations which are A-Res are also P-Ram and mutations which are A-Ram are also P-Res, that when an A-site Ram antibiotic is combined with a P-site RAM antibiotic growth is restored, and that A-Ram antibiotics have a P-Res effect on P-site tRNA affinity. These data cause us to conclude that the reciprocation model is a valid model for translation and demonstrates the interdependence of the P-site and A-site for decoding accuracy.