The Polypeptide Tunnel System in the Ribosome and Its Gating in Erythromycin Resistance Mutants of L4 and L22

Wadsworth Center, State University of New York at Albany, P.O. Box 509, Albany, NY 12201, USA.
Molecular Cell (Impact Factor: 14.02). 08/2001; 8(1):181-8. DOI: 10.1016/S1097-2765(01)00293-3
Source: PubMed


Variations in the inner ribosomal landscape determining the topology of nascent protein transport have been studied by three-dimensional cryo-electron microscopy of erythromycin-resistant Escherichia coli 70S ribosomes. Significant differences in the mouth of the 50S subunit tunnel system visualized in the present study support a simple steric-hindrance explanation for the action of the drug. Examination of ribosomes in different functional states suggests that opening and closing of the main tunnel are dynamic features of the large subunit, possibly accompanied by changes in the L7/L12 stalk region. The existence and dynamic behavior of side tunnels suggest that ribosomal proteins L4 and L22 might be involved in the regulation of a multiple exit system facilitating cotranslational processing (or folding or directing) of nascent proteins.

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    • "It is comprised primarily of 23S rRNA along with four ribosomal proteins (r-proteins), namely L4, L22, L23, and L29. Of these, L4 and L22 are the best characterized owing to their involvement in resistance to macrolides such as erythromycin [2]. Recently , however, much attention has been given to r-proteins L23 and L29 owing to their close proximity to the exit site where newly formed polypeptides emerge. "
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    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.
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    • "This is because RPL4 is a structural constituent of the large ribosomal subunit and covers the tunnel region to which these antibiotics bind. Mutation in RPL4 induces narrowing of the tunnel entrance, such that the antibiotics can no longer bind (Gabashvili et al., 2001). Furthermore, mild resistance to streptomycin is expected as both RPS12 and RPL4 are located in close proximity to the universally conserved adenine of the acceptor site (A-site) of the ribosome, A2451, in bacteria (Rakauskaite and Dinman, 2008). "
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    • "In one scenario, EspP 1-25 would transmit a signal (that is disrupted by either cis-acting point mutations or the trans-acting L22 mutation) that leads to the dissociation of TF from ribosomes. In another scenario, EspP 1-25 would direct the protein to one of the " alternate " ribosome tunnels whose existence was suggested by cryo-EM studies (Gabashvili et al., 2001) and the nascent chain would never encounter TF. In either case EspP 1-25 -MetE might aggregate when it is overproduced because the excess protein overwhelms the folding capacity of DnaK. "
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    ABSTRACT: In this report, we describe insights into the function of the ribosome tunnel that were obtained through an analysis of an unusual 25 residue N-terminal motif (EspP(1-25) ) associated with the signal peptide of the Escherichia coli EspP protein. It was previously shown that EspP(1-25) inhibits signal peptide recognition by the signal recognition particle, and we now show that fusion of EspP(1-25) to a cytoplasmic protein causes it to aggregate. We obtained two lines of evidence that both of these effects are attributable to the conformation of EspP(1-25) inside the ribosome tunnel. First, we found that mutations in EspP(1-25) that abolished its effects on protein targeting and protein folding altered the cross-linking of short nascent chains to ribosomal components. Second, we found that a mutation in L22 that distorts the tunnel mimicked the effects of the EspP(1-25) mutations on protein biogenesis. Our results provide evidence that the conformation of a polypeptide inside the ribosome tunnel can influence protein folding under physiological conditions and suggest that ribosomal mutations might increase the solubility of at least some aggregation-prone proteins produced in E. coli.
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