A non-radioactive method to detect and measure 48S initiation complex and 80S ribosome formation in vitro

Canonical eukaryotic translation initiation is cap- and scanning-dependent and is highly regulated by multiple factors, including cis-elements and trans-acting factors within the 5' untranslated region (UTR). Biochemically defining how different sequences and mutations in 5' UTRs affect initiation is critical to understanding the vastly different initiation rates among cellular mRNAs and how disease-linked mutations affect protein synthesis. Here, we provide details for an approach to detect and measure the abundance of 48S initiation complexes and 80S ribosomes on in vitro transcribed mRNAs using a commercially available rabbit reticulocyte lysate, along with the non-hydrolyzable GTP analog, GMPPNP, and the elongation inhibitor, cycloheximide. The outlined method relies on tracking the sedimentation of an experimental mRNA with each complex in sucrose density gradients by RT-qPCR, bypassing the need for radiolabeling mRNAs and scintillation counting. The protocol herein serves as a starting point for assessing translation initiation at distinct biochemical stages in vitro.