Versatility of trigger factor interactions with ribosome-nascent chain complexes.

Area of Science:

• Molecular biology
• Protein folding
• Bacterial protein synthesis

Background:

• Trigger factor (TF) is a primary molecular chaperone interacting with nascent polypeptide chains during bacterial protein synthesis.
• TF is composed of N-terminal ribosome binding, PPIase, and C-terminal domains involved in polypeptide interaction.

Purpose of the Study:

• To directly monitor the interactions of TF with nascent polypeptide chains using site-specifically labeled TF variants.
• To investigate the binding mechanisms and substrate specificity of TF.

Main Methods:

• Site-specific labeling of TF variants with an NBD fluorophore for fluorescence monitoring.
• Translation of firefly luciferase and other proteins to study TF-nascent chain interactions.
• Classification of the E. coli proteome based on predicted TF interaction strength.

Main Results:

• TF fluorescence increased significantly with hydrophobic nascent chains (e.g., luciferase) but not with less hydrophobic chains.
• TF residence time on nascent chains (111 ± 7 s) was much longer than on ribosomes (t(1/2) ≈ 10–14 s), indicating multiple TF molecules can bind per chain.
• TF binding correlated with hydrophobic regions and the ability of nascent chains to bury hydrophobicity, but also recognized discontinuous hydrophobic features and bound a folded protein domain, suggesting diverse mechanisms.

Conclusions:

• TF interacts with nascent polypeptide chains through multiple mechanisms, including recognition of linear and discontinuous hydrophobic segments, and binding to folded protein domains.
• TF exhibits broad substrate specificity, interacting with a wide range of proteins beyond just hydrophobic nascent chains.
• TF's extended residence time on nascent chains suggests a crucial role in early protein folding and quality control in bacteria.