Versatility of Trigger Factor Interactions with Ribosome-Nascent Chain Complexes

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 (t1 ⁄ 2 ϳ 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.

Interplay between trigger factor and other protein biogenesis factors on the ribosome

“…The dissociation of TF from Lep75-RNC gave rise to a single-exponential time course (k off E12 s À 1 ), as with non-translating ribosomes, indicating that an interaction of TF with the nascent Lep peptide, although it comprises a hydrophobic element, does not contribute significantly to the stability of the complex, again in accordance with the equilibrium titration. Comparable observations have been made previously with RNCs comprising various hydrophobic sequence elements, although half-life times of RNC-TF complexes generally were much longer 5 .…”

“…To study the influence of nascent chains with or without TF-specific sequences on the interaction of TF with RNCs, we have used MDCC-labelled RNCs carrying various nascent chains. The length of the nascent chain was restricted to 75 amino acids to avoid TF binding to longer nascent chains off the ribosome 5 . The affinities of TF for binding to those RNCs we determined by equilibrium titrations (Supplementary Fig.…”

“…TF is present in the cell in excess over ribosomes and potentially interacts with many nascent proteins 4,5 , with a preference for hydrophobic stretches flanked by positively charged amino acids 6 . Structural and mechanistic aspects of TF function have been reviewed recently 4,7,8 .…”

Firefly luciferase mutants as sensors of proteome stress

“…TF can also recognize hydrophilic surfaces on certain folded domains of ribosomal protein S7 (Lakshmipathy et al, 2010). A recent study on the functions of TF suggests that bacterial outer membrane proteins are the most prominent substrates of TF and loss of TF results in premature, co-translational protein translocation (Oh et al, 2011).…”

FKBPs in bacterial infections

“…Hence, multiple TF proteins assist during maturation of newly translated proteins (Hoffmann et al, 2010;Kramer et al, 2004;Lakshmipathy et al, 2010;Saio et al, 2014). Since, during translation nascent polypeptides exit the ribosomes in an unfolded state with isomerization prone peptidyl-prolyl bonds, the participation of TF has initially been considered as the ultimate proof for an in vivo PPIase function.…”