Timing and specificity of cotranslational nascent protein modification in bacteria

Abstract

The nascent polypeptide exit site of the ribosome is a crowded environment where multiple ribosome-associated protein biogenesis factors (RPBs) compete for the nascent polypeptide to influence their localization, folding, or quality control. Here we address how N-terminal methionine excision (NME), a ubiquitous process crucial for the maturation of over 50% of the bacterial proteome, occurs in a timely and selective manner in this crowded environment. In bacteria, NME is mediated by 2 essential enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). We show that the reaction of MAP on ribosome-bound nascent chains approaches diffusion-limited rates, allowing immediate methionine excision of optimal substrates after deformylation. Specificity is achieved by kinetic competition of NME with translation elongation and by regulation from other RPBs, which selectively narrow the processing time window for suboptimal substrates. A mathematical model derived from the data accurately predicts cotranslational NME efficiency in the cytosol. Our results demonstrate how a fundamental enzymatic activity is reshaped by its associated macromolecular environment to optimize both efficiency and selectivity, and provides a platform to study other cotranslational protein biogenesis pathways.

A ribosome-associated chaperone enables substrate triage in a cotranslational protein targeting complex

“…8 , right panel). As shown here and previously for prokaryotic SRP 52 , 66 and N-terminal methionine excision enzymes 42 , the balance between efficiency and specificity of a cotranslational protein biogenesis pathway can be significantly reshaped by translation elongation and spatiotemporal coordination with other RPBs at the ribosome exit site. Our work provides a valuable conceptual framework, as well as experimental tools, to investigate this coordination on the mammalian ribosome at energetic and molecular detail, which can be readily extended to other cotranslational protein biogenesis pathways.…”

“…Kinetic modeling emphasizes the role of NAC in the specificity of cotranslational protein targeting. To quantitatively understand how the NAC-induced regulation of SRP observed in our reconstituted system impact this pathway under in vivo-like conditions, we constructed an analytical kinetic model for cotranslational protein targeting by SRP 42 (Fig. 7b).…”

Regulation of ribosomal protein genes: An ordered anarchy

“…Phosphorylation can also alter ribosome biogenesis or even lead to extra ribosomal functions. These protein modifications may occur either co‐translationally or post‐translationally, and they are considered the best source for a potential ribosome specialization (Genuth & Barna, 2018b; Ruiz‐Canada, Kelleher, & Gilmore, 2009; Sauert, Temmel, & Moll, 2015; C. I. Yang, Hsieh, & Shan, 2019). In the following section, we will discuss the impact of specific modifications on RP regulation, functions, and stability.…”

Mechanisms of Cotranslational Protein Maturation in Bacteria

“…for further methionine excision by the methionine aminopeptidase (MAP) (Yang et al, 2019). Both of these enzymes bind near the exit of the ribosomal tunnel (Bingel-Erlenmeyer et al, 2008;Sandikci et al, 2013) and while an excess of one factor reduces the binding of the other, a recent structural study suggested that MAP may reposition itself to a secondary binding site if excess of PDF is present (Bhakta et al, 2019).…”