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Bacterial RF3 Senses Chaperone Function in Co-translational Folding

ABSTRACT: Molecular chaperones assist in protein folding by interacting with nascent polypeptide chains (NCs) during translation, but whether the ribosome can sense chaperone defects and abort translation of misfolding NCs has not been explored. Here we used quantitative proteomics in E. coli to investigate the ribosome-associated chaperone network 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 of NCs due to defects in co-translational chaperone function or amino acid analog incorporation, results in recruitment of the non-canonical release factor RF3 to the ribosome. RF3 then cooperates with RF2 in mediating premature chain termination of a subset of abundant NCs, allowing their efficient degradation. This function of RF3 reduces the accumulation of misfolded proteins. It is critical for proteostasis maintenance and cell growth under conditions of limited chaperone availability.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Escherichia Coli

SUBMITTER: Roman Körner

LAB HEAD: F. Ulrich Hartl

PROVIDER: PXD025219 | Pride | 2022-12-09

REPOSITORIES: Pride

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Project description:Transcriptome profiles were analyzed using the samples taken at the exponential and stationary phases during the cultivation of REL606 and MG1655 in LB medium. At the exponential growth phase, most highly expressed genes of B were those for replication, translation, or nucleotide transport and metabolism, while many of the K-12 genes were involved in cell motility, transcription, carbohydrate transport, or energy production. At the stationary phase, many of the genes highly expressed in B were for transport and metabolism of various amino acids and carbohydrates, whereas those in K-12 had functions related to cell motility, ribosomal subunit protein production, or energy generation. Many genes in REL606 and MG1655 showed highly distinct expression levels irrespective of growth conditions. Highly expressed genes in REL606 included those encoding enzymes for biosynthesis of L-arginine (argAGDECBHI) and branched-chain amino acid (ilvGMEDA), and those encoding a subunit of the L-arginine transporter (artJ), cytochrome b562 (cybC), subunits of the histidine ABC transporter (hisPJ), cytotoxins (hokED), outer membrane porin (ompF), L-arginine decarboxylase (speA), and cell division inhibitor (sulA). Highly expressed genes in MG1655 included those for chemotaxis (cheZYRWA, tap, trg, tsr), Lon protease (lon), C4-dicarboxylate-sensing histidine kinase (dcuS), chaperones (clpB, dnaK, groES, htpG, ibpA), the major subunit of type 1 fimbriae (fimA), a regulator of flagellar biosynthesis (flhC), glycerol-3-phosphate-dehydrogenase (glpABCD), glycerophosphoryl diester phosphodiesterase (glpQ), glycerol-3-phosphate transporter (glpT), hydrogenase 2 (hybCBO), outer membrane porins (nmpC, ompA, ompC), and galactitol transport and metabolism (gatYZC). All microarray experiments were performed in duplicate with dye-swapping. The probes were spotted in duplicate onto the microarray. Thus, the log2 of the intensity ratio with the two dyes for each spot was calculated from up to four values from the duplicated spot on the microarray and the dye-swap experiment.

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Bacterial RF3 Senses Chaperone Function in Co-translational Folding

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