Molecular chaperones assist with protein folding by interacting with nascent polypeptide chains (NCs) during translation. Whether the ribosome can sense chaperone defects and, in response, abort translation of misfolding NCs has not yet been explored. Here we used quantitative proteomics to investigate the ribosome-associated chaperone network in E. coli and the consequences of its dysfunction. Trigger factor and the DnaK (Hsp70) system are the major NC-binding chaperones. HtpG (Hsp90), GroEL, and ClpB contribute increasingly when DnaK is deficient. Surprisingly, misfolding because of defects in co-translational chaperone function or amino acid analog incorporation results in recruitment of the non-canonical release factor RF3. RF3 recognizes aberrant NCs and then moves to the peptidyltransferase site to cooperate with RF2 in mediating chain termination, facilitating clearance by degradation. This function of RF3 reduces the accumulation of misfolded proteins and is critical for proteostasis maintenance and cell survival under conditions of limited chaperone availability. [Display omitted] • Proteomics analysis reveals the co-translational chaperone network of E. coli • Defective chaperone binding to nascent chains results in recruitment of RF3 • RF3 mediates premature termination of misfolded nascent polypeptides • RF3 is critical for survival of E. coli under conditions of limited chaperone capacity Chaperone-defective E. coli cells can abort translation of misfolded polypeptides. The non-canonical release factor RF3 recognizes aberrant nascent chains and is recruited to the ribosome, where it cooperates with RF2 in terminating translation. This function is critical for proteostasis maintenance. [ABSTRACT FROM AUTHOR]