Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes

Lorna E Lancaster,Colin Kleanthous,Andreas Savelsbergh,Wolfgang Wintermeyer,Marina V Rodnina

doi:10.1111/j.1365-2958.2008.06283.x

Abstract

The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon–anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon–recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA–mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin.

Highlights

During times of stress, some strains of Escherichia coli produce the plasmid-encoded cytotoxin colicin E3
We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis
We find that the cleavage affects predominantly the elongation step

Summary

Introduction

Some strains of Escherichia coli produce the plasmid-encoded cytotoxin colicin E3. It is a ~60 kDa protein with three domains that are required for the toxin to enter and kill the cell. The receptor binding domain binds to the outer membrane protein, BtuB, of competing E. coli (Imajoh et al, 1982), and the translocation domain interacts with the Tol complex of proteins involved in the maintenance of outer membrane stability (Garinot-Schneider et al, 1997; Bouveret et al, 1998). The cytotoxic domain interacts directly with the bacterial inner membrane and is transported to the cytoplasm, possibly through the action of FtsH (Mosbahi et al, 2004; Walker et al, 2007). Im3 forms a complex with the cytotoxic domain of colicin E3 with a binding affinity of 10-12 M and inactivates the RNase (Jakes and Zinder, 1974a; Kleanthous and Walker, 2001; Walker et al, 2003)

Results

Discussion

Conclusion