Regulation of mRNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by the mRNA untranslated regions (UTRs) and often target translation initiation. Initiation consists of dynamic interactions between eukaryotic initiation factors (eIFs) and the small ribosomal subunit (SSU) and concludes with joining of the large ribosomal subunit (LSU) to form a full ribosome at the start codons of mRNA. However, no methods existed to study the mechanistic intricacies and regulation of translation initiation transcriptome-wide.

To address this, we developed translation complex profile sequencing (TCP-seq)¹, a method related to the ribosome profiling approach. TCP-seq is uniquely capable of recording positions of any type of ribosome-mRNA complex across all RNA. It uses fast covalent fixation of translation complexes in live cells, followed by RNase footprinting of translation intermediates and their separation into full ribosome and SSU fractions. Ribosome and SSU-derived footprints are then deep sequenced separately, generating near-native complex distribution profiles during the initiation, elongation, termination and recycling stages of translation.

TCP-seq use was highly successful in discerning long-standing mechanistic questions of translation in yeast model system, including resolving the elusive ‘scanning’ process, visualizing start codon recognition events and capturing diverse conformations of elongating ribosomes¹. Here we outline further improvements to TCP-seq that make it a versatile, flexible approach which can be used to study regulation of protein synthesis with unprecedented depth of detail. Combined with yeast strains with altered activity of eIFs, it can be used to resolve differential initiation mechanisms utilised across transcripts and pinpoint regulatory elements in 5'UTRs of mRNA. Used in combination with affinity binding (co-immunoprecipitation), TCP-seq can provide a snapshot of all translation events that involve a certain protein factor and/or cellular location. Adapted to perform on mammalian cells, TCP-seq can be used to identify mechanisms of protein synthesis control active in biologically important microenvironments, such as nutrient deficiency, ageing and development of cancer, and thus provide entirely new perspectives on the development of better treatments.