Selective Ribosome Profiling Reveals the Cotranslational Chaperone Action of Trigger Factor In Vivo

Abstract

SUMMARY As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting and folding factors. Here we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone Trigger Factor (TF) reveal that, while TF can interact with many polypeptides, β-barrel outer membrane proteins are the most prominent substrates. Loss of TF leads to broad outer membrane defects and premature, cotranslational protein translocation. While in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotrantslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting ad folding factors.

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

“…Whereas PDF can act on ribosomes exposing short nascent chains independent of the sequence 1 , or even on isolated short oligopeptides, TF binding to RNCs with short nascent chains is transient, and the complex is stabilized when nascent chains containing TF-specific hydrophobic patches become available. Thus, PDF and TF can act sequentially, in accordance with the observed rapid kinetics of PDF binding to ribosomes 28 and the binding preference of TF for RNCs carrying nascent peptides of about 100 amino acids or longer observed by directed ribosome profiling in vivo 27 .…”

“…Deformylation and methionine removal occur on 60-80% of all proteins and control the stability of proteins, as the N-terminal amino acid determines the susceptibility of many mature proteins for the cellular protein degradation machinery 25,26 . It has been proposed that PDF and TF act in a concerted fashion 3 , whereas recent data from selective ribosome profiling indicated that TF and PDF do compete for ribosome binding in vivo 27 . Conversely, on the basis of binding data obtained in vitro, it was suggested that TF and SRP did not compete with PDF for binding to ribosomes or RNCs 28 .…”

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). On the basis of these findings, similar functions of NAC and RAC can be envisioned.…”

Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

“…Accumulating data indicate that this process begins at the ribosome exit site, where many protein biogenesis machineries can interact and gain access to the nascent polypeptide. This includes chaperones (1-5) such as trigger factor (TF) (1,4,6,7), Hsp70, and the nascent polypeptide-associated complex (8)(9)(10)(11)(12)(13); modification enzymes (10,(14)(15)(16) such as N-acetyl transferase, methionine aminopeptidase, and arginyl transferase; protein-targeting and translocation machineries such as signal recognition particle (SRP) (17)(18)(19)(20), SecA (21), the SecYEG (or Sec61p) (22,23) and YidC translocases (24,25), and the ribosome-bound quality control complex (26)(27)(28)(29)(30). Engagement of these factors with nascent polypeptides influences their folding, assembly, localization, processing, and quality control.…”