In vitro reconstitution and biochemical characterization of translation initiation by internal ribosomal entry

The internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV) and related viruses promote initiation of translation by a noncanonical end-independent mechanism. To characterize this mechanism at the molecular level, we have developed biochemical approaches to reconstitute the process in vitro from individual purified components of the translation apparatus, developed methods to characterize steps in this process so that the functions of individual proteins can be characterized, and adapted assays such as primer extension inhibition ("toe printing") to monitor accurate assembly on the IRES of ribosomal 48S and 80S complexes. In vitro reconstitution of 48S complex formation offers an approach for the functional identification of IRES trans-acting factors (ITAFs) that are required for initiation in addition to canonical initiation factors and revealed that despite being related, different EMCV-like IRESs nevertheless have distinct ITAF requirements. Toe printing revealed that a common feature of initiation on EMCV-like IRESs is the stable binding of an eIF4G/eIF4A complex to them near the initiation codon, where it can locally unwind RNA to facilitate ribosomal attachment. The same toe printing assay indicated that binding of ITAFs to these IRESs enhances binding of these two canonical initiation factors. We also describe protocols for chemical and enzymatic footprinting to determine the interactions of trans-acting factors with the IRES at nucleotide resolution and for directed hydroxyl radical probing to determine their orientation on the IRES.