Abstract
Trigger factor (TF) is the first molecular chaperone that interacts with nascent chains emerging from bacterial ribosomes. TF is a modular protein, consisting of an N-terminal ribosome binding domain, a PPIase domain, and a C-terminal domain, all of which participate in polypeptide binding. To directly monitor the interactions of TF with nascent polypeptide chains, TF variants were site-specifically labeled with an environmentally sensitive NBD fluorophore. We found a marked increase in TF-NBD fluorescence during translation of firefly luciferase (Luc) chains, which expose substantial regions of hydrophobicity, but not with nascent chains lacking extensive hydrophobic segments. TF remained associated with Luc nascent chains for 111 +/- 7 s, much longer than it remained bound to the ribosomes (t((1/2)) approximately 10-14 s). Thus, multiple TF molecules can bind per nascent chain during translation. The Escherichia coli cytosolic proteome was classified into predicted weak and strong interactors for TF, based on the occurrence of continuous hydrophobic segments in the primary sequence. The residence time of TF on the nascent chain generally correlated with the presence of hydrophobic regions and the capacity of nascent chains to bury hydrophobicity. Interestingly, TF bound the signal sequence of a secretory protein, pOmpA, but not the hydrophobic signal anchor sequence of the inner membrane protein FtsQ. On the other hand, proteins lacking linear hydrophobic segments also recruited TF, suggesting that TF can recognize hydrophobic surface features discontinuous in sequence. Moreover, TF retained significant affinity for the folded domain of the positively charged, ribosomal protein S7, indicative of an alternative mode of TF action. Thus, unlike other chaperones, TF appears to employ multiple mechanisms to interact with a wide range of substrate proteins.
Highlights
Trigger factor (TF)3 binds to the large ribosomal subunit close to the polypeptide exit site [6] and has been shown by chemical cross-linking to interact with a wide variety of nascent polypeptide chains [7,8,9,10,11,12]
Monitoring the Interaction of TF with Nascent Chains during Translation—TF is the first chaperone to interact with nascent chains as they emerge from the ribosomal exit tunnel
Luc was initially chosen as a model substrate for these experiments, based on previous observations indicating the high affinity of TF for Luc ribosome-nascent chain complexes (RNCs) [16, 17, 34]
Summary
Trigger factor (TF)3 binds to the large ribosomal subunit close to the polypeptide exit site [6] and has been shown by chemical cross-linking to interact with a wide variety of nascent polypeptide chains [7,8,9,10,11,12]. Was present during translation, no significant increase in NBD fluorescence was observed (Fig. 1C), confirming that ribosome binding is a prerequisite for the interaction of TF with nascent chains.
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