Proteins à la carte: riboproteogenomic exploration of bacterial N-terminal proteoform expression.

Abstract

With the emerging theme of genes within genes comprising the existence of alternative open reading frames (ORFs) generated by translation initiation at in-frame start codons, mechanisms that control the relative utilization of annotated and alternative TIS need to be unraveled and our molecular understanding of resulting proteoforms broadened. Utilizing complementary ribosome profiling strategies to map ORF boundaries, we uncovered dual-encoding ORFs generated by in-frame TIS usage in Salmonella. Besides demonstrating that alternative TIS usage may generate proteoforms with different characteristics, such as differential localization and specialized function, quantitative aspects of conditional retapamulin-assisted ribosome profiling (Ribo-RET) translation initiation maps offer unprecedented insights into the relative utilization of annotated and alternative TIS, enabling the exploration of gene regulatory mechanisms that control TIS usage and, consequently, the translation of N-terminal proteoform pairs.