Identification of Nascent Chain Interaction Sites on Trigger Factor

Sathish K Lakshmipathy,Sladjana Tomic,Christian M Kaiser,Hung-Chun Chang,Pierre Genevaux,Costa Georgopoulos,José M Barral,José M Barral,Arthur E Johnson,Arthur E Johnson,Arthur E Johnson,F Ulrich Hartl,Stephanie A Etchells

doi:10.1074/jbc.m609871200

Abstract

The role of ribosome-binding molecular chaperones in protein folding is not yet well understood. Trigger factor (TF) is the first chaperone to interact with nascent polypeptides as they emerge from the bacterial ribosome. It binds to the ribosome as a monomer but forms dimers in free solution. Based on recent crystal structures, TF has an elongated shape, with the peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and the N-terminal ribosome binding domain positioned at opposite ends of the molecule and the C-terminal domain, which forms two arms, positioned in between. By using site specifically labeled TF proteins, we have demonstrated that all three domains of TF interact with nascent chains during translation. Interactions with the PPIase domain were length-dependent but independent of PPIase activity. Interestingly, with free TF, these same sites were found to be involved in forming the dimer interface, suggesting that dimerization partially occludes TF-nascent chain binding sites. Our data indicate the existence of two regions on TF along which nascent chains can interact, the NC-domains as the main site and the PPIase domain as an auxiliary site.

Highlights

Possible at lower temperature, and the resulting ⌬tig⌬dnaK cells can be adapted to growth at up to 30 °C [4, 5]
In the recent crystal structures, Trigger factor (TF) displays an elongated shape [12, 14] in which the PPIase domain is connected with the N-domain via a long extension, so that it is positioned at the other end of the molecule, whereas the C-domain lies between the N-domain and the PPIase domain (Fig. 1A)
Characterization of TF with Photoreactive Probes at Different Locations—To determine which regions of TF interact with the nascent chain as it emerges from the ribosome, we made use of a recently developed technology [32] allowing for in vivo expression of a protein with a benzophenone photoreactive probe incorporated at a specific site through amber codon suppression

Summary

Introduction

Possible at lower temperature, and the resulting ⌬tig⌬dnaK cells can be adapted to growth at up to 30 °C [4, 5]. To determine which domain(s) of TF interact with elongating nascent polypeptides, we placed photoreactive probes at specific sites in each domain of TF and added the purified probe containing proteins to a reconstituted in vitro translation system essentially free of chaperones [15, 31]. Characterization of TF with Photoreactive Probes at Different Locations—To determine which regions of TF interact with the nascent chain as it emerges from the ribosome, we made use of a recently developed technology [32] allowing for in vivo expression of a protein with a benzophenone photoreactive probe incorporated at a specific site through amber codon suppression.

Results

Conclusion