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

Abstract

SummaryThe 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.

The ribosome as a molecular machine: the mechanism of tRNA–mRNA movement in translocation

“…GTP hydrolysis drives a rearrangement of the ribosome (referred to as unlocking) that precedes and limits the rate of tRNA-mRNA movement on the 30S subunit [20]. The structural basis for unlocking is not known, as a structural model of this transient ribosome-EF-G complex is not available, but it is likely to involve a change in the mobility of elements of the 30S subunit, possibly by facilitating the swivelling movement of the 30S head [27,28]. GTP hydrolysis by EF-G is coupled to the acceleration of unlocking [20,29], but the mechanism of coupling between GTP hydrolysis and the conformational changes in the complex is unknown.…”

Bacteriocins and their position in the next wave of conventional antibiotics

“…In short, it cleaves stop codons into the A site. It decreases the stability of the codon recognition complex, slowing aminoacyl-tRNA accommodation at the A site [18,54]. A c c e p t e d M a n u s c r i p t tRNA Tyr > tRNA Asp > tRNA His > tRNA Asn [20].…”

“…Members of the E-group endonuclease colicins have the H-N-H motif and bind the BtuB/Tol translocation machinery in order to cross the outer membrane [56,57]. These bacteriocins cleave the 16S rRNA at the 3'-end of the coding sequence, which inhibits translation [18,54,57]. It does so by accumulation of sequential impaired decoding events that results in low occupancy at the A site and inability to elongate the peptide past the first few codons.…”

“…In addition, bacteriocins >10 kDa are parsed into a third class [12]. Bacteriocins from Gram-negative bacteria are divided [16,17] into small peptides, such as microcins, and large peptides, such as colicins [18][19][20]. Further subdivisions exist within these broader categories, including instances of homology in motifs [21].…”

Ribosomal Encoded Bacteriocins: Their Functional Insight and Applications

“…Most of these nuclease bacteriocins elicit cell death through either hydrolases or transferases by targeting phosphodiester bonds in the bacterial cytoplasm [31]. They target either genomic DNA (DNases), 16S rRNA (rRNases), or tRNAs (tRNases) and begin their passage into cells through either the Tol or Ton system [32][33][34]. After translocation of nuclease bacteriocin across the OM and periplasm, cytotoxic domain behaves differently from that of the pore-forming colicins, as the entire domain has to cross the cytoplasmic membrane.…”