Mechanism and Function of Nascent Protein Modification in Bacteria

Author: Yang, Chien-I

Year: 2023

Degree: Dissertation (Ph.D.)

Advisor: Shan, Shu-ou

Committee Members: Dougherty, Dennis A.; Hsieh-Wilson, Linda C.; Varshavsky, Alexander J.; Shan, Shu-ou

Option: Chemistry

DOI: 10.7907/qhz7-a383

Abstract

Newly synthesized proteins undergo multiple modifications to ensure proper biogenesis and acquire their functions. N-terminal methionine excision (NME), mediated by the sequential actions of peptide deformylase (PDF) and methionine aminopeptidase (MAP), is an essential and the most prevalent N-terminal protein modification in the bacterial proteome. Despite the extensive studies on enzymatic catalysis, how NME impacts various cellular functions and how the enzymes achieve timing and selectivity under complex cellular conditions have been long-standing puzzles.

In this work, we use a combination of biochemical analyses, computational modeling, and in vivo measurements to investigate the molecular mechanisms and physiological functions of cotranslational NME reactions. We show that the interactions between the ribosome, the nascent chain, the NME enzymes, and other ribosome-associated protein biogenesis factors dramatically remodel the kinetics and specificity of NME reactions under physiological conditions. In addition, we apply time-resolved, system-wide analyses on the translatome and steady-state proteome to study how the inhibition of PDF influences diverse cellular pathways in bacteria. The results unveil the impact of NME on the biogenesis of nascent proteins and highlight the role of the membrane in coupling the biochemical activities of NME enzymes to cellular physiology.

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