Trigger Factor Forms a Protective Shield for Nascent Polypeptides at the Ribosome

Abstract

In prokaryotes, the ribosome-associated Trigger Factor is the first chaperone newly synthesized polypeptides encounter when they emerge from the ribosomal exit tunnel. The effects that Trigger Factor exerts on nascent polypeptides, however, remain unclear. Here we analyzed the potential of the Trigger Factor to shield nascent polypeptides at the ribosome. A set of arrested nascent polypeptides differing in origin, size, and folding status were synthesized in an Escherichia coli-based in vitro transcription/translation system and tested for sensitivity to degradation by the unspecific protease proteinase K. In the absence of Trigger Factor, nascent polypeptides exposed outside the ribosomal exit tunnel were rapidly degraded unless they were folded into a compact domain. The presence of Trigger Factor, as well as a Trigger Factor fragment lacking its peptidyl-prolyl isomerase domain, counteracted degradation of all unfolded nascent polypeptides tested. This protective function was specific for ribosome-tethered Trigger Factor, since neither non-ribosomal Trigger Factor nor the DnaK system, which cooperates with Trigger Factor in the folding process in vivo, revealed a comparable efficiency in protection. Furthermore, shielding by Trigger Factor was not restricted to short stretches of nascent chains but was evident for large, non-native nascent polypeptides exposing up to 41 kDa outside the ribosome. We suggest that Trigger Factor supports productive de novo folding by shielding nascent polypeptides on the ribosome thereby preventing untimely degradation or aggregation processes. This protected environment provided by Trigger Factor might be particularly important for large multidomain proteins to fold productively into their native states.Cells employ a large arsenal of molecular chaperones to promote the folding of newly synthesized proteins in the cytosol (1-3). At the forefront are chaperones, which transiently bind to ribosomes and thereby are optimally positioned to associate with emerging nascent polypeptides. In eubacteria, Trigger Factor (TF) 3 binds to ribosomes near the exit of the peptide tunnel through major contacts to L23 (4 -7) and interacts co-translationally with nascent polypeptides (8, 9). Downstream of TF, the DnaK-DnaJ-GrpE (referred to as KJE) and GroELGroES chaperone systems assist further folding steps to the native state (2, 10). Together these chaperones form a folding network of considerable robustness. Thus, the lack of TF can be compensated by activity of the KJE system, while simultaneous deletion of dnaK and the TF encoding tig gene results in synthetic lethality at Ն30°C and massive protein aggregation (11-13).TF is a three-domain protein of 48 kDa that adopts an unusual extended conformation (Fig. 1A) (5, 14). The N-terminal domain builds the "tail" of the molecule and harbors the "TF-signature motif," which mediates ribosome docking (4). A peptidyl-prolyl cis/trans isomerase (PPIase) domain with structural homology to FK506-binding proteins (9, 16) is located at the other end of ...

Dynamics of Trigger Factor Interaction with Translating Ribosomes

“…We generated RNCs exposing, in addition to the triple strep tag, either the N-terminal 27, 66, 108, 177, or 318 aa of ICDH (ICDH-27 to ICDH-318) or the N-terminal 23, 36, 100, 148, 190, 233, 325, or 516 aa of RpoB (RpoB-23 to RpoB-516). None of the ICDH derived nascent chains should be capable of adopting a compactly folded structure as judged from its atomic structure (23) and the finding that several truncated ICDH nascent chains are highly protease-sensitive (18). For RpoB, the structural features of two homologs (24,25) suggest that RpoB-148 may fold into the first N-terminal RpoB domain and RpoB-516 encompasses the first two domains of RpoB.…”

“…To induce translational arrest, we genetically fused the 3Ј-end of the open reading frames of target genes to a linker encoding both the SecM peptide, which induces translational arrest (21), and additional amino acids (aa) to allow the complete exposure of the target protein at the ribosomal exit site (Fig. 1A) (18). In order to affinity purify the stalled RNCs, we introduced a triple strep tag sequence at the 5Ј-end of the gene construct (20).…”

“…Purification of Ribosomes and RNCs-Ribosomes were purified from strain MC4100 ⌬tig as described (18). RNCs used for kinetics measurements were purified from E. coli strain BL21(DE3) ⌬tig, which contained one of the arrested nascent chain plasmids (pBAT-Strep3-SecM) and the pZA4 plasmid.…”

Co-translational Binding of GroEL to Nascent Polypeptides Is Followed by Post-translational Encapsulation by GroES to Mediate Protein Folding

“…The prevailing view assumes that TF and the DnaK system act as co-translational chaperones to prevent protein aggregation, whereas GroEL acts as a post-translational chaperone to help polypeptides that have been released from the ribosome to reach their native state (4,19). This model of the roles of the major chaperones is essentially based on the well designed in vitro order-of-addition experiments (20), on genetic experiments showing that simultaneous deletion of TF and DnaK is lethal (9, 21), and on the fact that TF is a ribosome-tethered chaperone (22)(23)(24)(25)(26). However, the recent finding that overproduction of GroEL and GroES permits the growth of the E. coli lacking TF and DnaK (27, 28) has raised questions as to whether the roles of these chaperones are non-overlapping.…”

Correction

“…One suggestion is that, as well as preventing misfolding, trigger factor functions in a static manner by actively folding proteins within a confined void that is created when it binds to the ribosome 8 . This void has never been observed experimentally.…”