Initiation factor IF1 reveals mechanistic details for A and P-site decoding accuracy

Initiation factor IF1 is the least understood initiation factor to date. IF1 binds the A site of the ribosome during translation initiation. This dissertation presents clear evidence that IF1 influences the selection of initiator tRNA in the P site through the use of lacZ fusion reporters in vivo, and experiments in vitro. IF1's influence appears to be mediated via a functional mechanism in which decoding accuracy between the A and P sites are linked and inverse. This dissertation proposes that IF1 binds the A site to functionally mimic a cognate decoding event in the A site. This mimic results in the A site being positioned in a non-decoding conformation and inversely placing the P site in to a decoding conformation for initiation. Decoding accuracy was examined by lacZ reporters in vivo bearing different near cognate start codons and comparisons are drawn with the canonical cognate AUG start codon. Neutral alanine substitution mutations were made at arginine residues within IF1 in order to test the arginine residues' interaction with the decoding center of the A site. Wild-type and mutant IF1 proteins were then purified in order to test them with an in vitro initiation assay to confirm the in vivo findings. In vitro preinitiation complexes were formed and an assay was developed which is capable of detecting single molecule fluorescently labeled components of the initiation complex. Fluorescently labeled tRNA was followed for release from the P site in pre-initiation complexes. tRNA release was examined with decoding accuracy antibiotics at high concentrations to test the reciprocation model using A-site and P-site miscoding antibiotics. The data with antibiotics used in tRNA release assays is consistent with reciprocity between the two decoding centers. Evidence is presented for IF1's role in decoding accuracy, and site specific mutations in IF1 give strong support for the model of decoding accuracy reciprocation between the A and P sites.