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The ribosomal exit tunnel as a target for optimizing protein expression in Escherichia coli.

ABSTRACT: The folding of many cellular proteins occurs co-translationally immediately outside the ribosome exit tunnel, where ribosomal proteins and other associated factors coordinate the synthesis and folding of newly translated polypeptides. Here, we show that the large subunit protein L29, which forms part of the exit tunnel in Escherichia coli, is required for the productive synthesis of an array of structurally diverse recombinant proteins including the green fluorescent protein (GFP) and an intracellular single-chain Fv antibody. Surprisingly, the corresponding mRNA transcript level of these proteins was markedly less abundant in cells lacking L29, suggesting an unexpected regulatory mechanism that links defects in the exit tunnel to the expression of genetic information. To further highlight the importance of L29 in maintaining protein expression, we used mutagenesis and selection to obtain L29 variants that enhanced GFP expression. Overall, our results suggest that the ribosomal exit tunnel proteins may be key targets for optimizing the overproduction of active, structurally complex recombinant proteins in bacterial cells.

SUBMITTER: Contreras-Martinez LM

PROVIDER: S-EPMC3382190 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mutational changes in bacterial ribosomes often affect gene expression and consequently cellular fitness. Understanding how mutant ribosomes disrupt global gene expression is critical to determining key genetic factors that affect bacterial survival. Here, we describe gene expression and phenotypic changes presented in Escherichia coli cells carrying an uL22(K90D) mutant ribosomal protein, which displayed alterations during growth. Ribosome profiling analyses revealed reduced expression of operons involved in catabolism, indole production, and lysine-dependent acid resistance. In general, translation initiation of proximal genes in several of these affected operons was substantially reduced. These reductions in expression were accompanied by increases in the expression of acid-induced membrane proteins and chaperones, the glutamate-decarboxylase regulon, and the autoinducer-2 metabolic regulon. In agreement with these changes, uL22(K90D) mutant cells had higher glutamate decarboxylase activity, survived better in extremely acidic conditions, and generated more biofilm in static cultures compared to their parental strain. Our work demonstrates that a single mutation in a non-conserved residue of a ribosomal protein affects a substantial number of genes to alter pH resistance and the formation of biofilms. IMPORTANCE All newly synthesized proteins must pass through a channel in the ribosome named the exit tunnel before emerging into the cytoplasm, membrane, and other compartments. The structural characteristics of the tunnel could govern protein folding and gene expression in a species-specific manner but how the identity of tunnel elements influences gene expression is less well-understood. Our global transcriptomics and translatome profiling demonstrate that a single substitution in a non-conserved amino acid of the E. coli tunnel protein uL22 has a profound impact on catabolism, cellular signaling, and acid resistance systems. Consequently, cells bearing the uL22 mutant ribosomes had an increased ability to survive acidic conditions and form biofilms. This work reveals a previously unrecognized link between tunnel identity and bacterial stress adaptation involving pH response and biofilm formation.

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The ribosomal exit tunnel as a target for optimizing protein expression in Escherichia coli.

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