Timing and Specificity of Cotranslational Nascent Protein Modification in Bacteria

Abstract

Selection of newly‐synthesized proteins into correct protein biogenesis pathways is crucial for cellular homeostasis. The ubiquitous N‐terminal methionine excision (NME) process is mediated by peptide deformylase (PDF) and methionine aminopeptidase (MAP), two essential enzymes in bacteria. This reaction takes place near the nascent peptide exit site of the ribosome, where multiple ribosome‐associating protein biogenesis factors (RPBs) also compete for the access to the nascent chain. How NME achieves its efficiency and specificity at this crowded environment is unknown. Here, using kinetic measurements on purified ribosome‐nascent chain complexes, we show that the ribosome accelerates the MAP reaction for optimal substrates by 10²–10⁴ folds. Kinetic competition with translation elongation and selective regulation from other RPBs enhance the specificity of NME by narrowing the processing time window for reactions on suboptimal substrates. With the kinetic data, we constructed a mathematical model and accurately predicted the cotranslational NME efficiency in cytosol. Our data demonstrate how a fundamental enzymatic activity is reshaped by its associated macromolecular environment to optimize both efficiency and selectivity. Moreover, the remodeled MAP activity prompted us to develop a cotranslational assay to screen for MAP inhibitors in the physiological context of translating ribosome. The results explain the discrepancy between traditional peptide‐based assays and cellular data, providing a powerful tool for the development of antibacterial agents targeting the NME machineries.

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