PROJECT SUMMARY Changes in physiological stimuli reprogram the translation machinery to alter preferential recruitment of mRNAs to the ribosome and which start site on a mRNA is selected. Genome wide analysis using ribosome profiling has revealed far more extensive regulation of these events than previously appreciated, yet the mechanism of mRNA recruitment and its regulation remains poorly understood. This knowledge gap persists mainly because of difficulties in determining which intermediate step(s) in the initiation pathway function as kinetic checkpoints to control mRNA recruitment. To date, initiation pathway intermediates have largely been identified on the basis of their thermodynamic stability which must withstand traditional assays (e.g. sucrose gradients and immunoprecipitations). These approaches take minutes to hours to perform, and/or require cross-linking agents to stabilize them, but most intermediates prior to initiation codon selection occur on the sub-second to second time scale. To determine how mRNAs are selected for translation, one must develop and use assays that can precisely monitor the formation of pathway intermediates in real-time. To overcome this bottleneck and move the field forward, we have developed innovative ensemble and single-molecule fluorescence-based assays that can monitor the rate of mRNA recruitment to the ribosome in real-time. Our highly purified reconstituted system will enable us to successfully test and build models with which to understand mRNA recruitment and its regulation. Models that we generate will be tested using translation assays in cell-free extracts and intact cells. Our long-term objective is to understand the mechanism by which alterations in initiation factor availability and their post-translational modification reprograms the translational apparatus to control which mRNAs are translated in response to physiological stimuli.