Figure 4: Interactions of the ErmBL nascent chain with the ribosome.

(a) Interactions of ErmBL (teal) with 23S rRNA (blue) within the exit tunnel of the ribosome. (b) Local resolution map of view shown in (a). (c) Alanine-scanning mutagenesis of ErmBL and effect of mutant peptides on ribosome stalling (arrowed) in the presence (+) and absence (−) of erythromycin (ERY) as determined by toe-printing. (d) Toe-printing of wild type (wt) and mutant (R7A) ErmBL on E. coli ribosomes with wt or mutated 23S rRNA nucleotide 2586, in the presence (+) or absence (−) of ERY. All the reactions contained borrelidin, a Thr-tRNA synthetase inhibitor, which, in the absence of ERY-dependent arrest at the Asp (D10) codon, causes translation arrest at the Lys (K11) codon. (e) Influence of the U2586A mutation on stalling efficiency with the wt and mutant ErmBL peptides (an average of two independent experiments; error bars represent the standard deviation of the mean). (f) The mutations of A2062 do not affect ErmBL stalling, nor rescue stalling impaired by the R7A mutation.