The key function of a conserved and modified rRNA residue in the ribosomal response to the nascent peptide

Abstract

The ribosome is able to monitor the structure of the nascent peptide and can stall in response to specific peptide sequences. Such programmed stalling is used for the regulation of gene expression. The molecular mechanisms of the nascent‐peptide recognition and ribosome stalling are unknown. We identified the conserved and posttranscriptionally modified 23S rRNA nucleotide m2A2503 located at the entrance of the ribosome exit tunnel as a key component of the ribosomal response mechanism. A2503 mutations abolish nascent‐peptide‐dependent stalling at the leader cistrons of several inducible antibiotic resistance genes and at the secM regulatory gene. Remarkably, lack of the C2 methylation of A2503 significantly function induction of expression of the ermC gene, indicating that the functional role of posttranscriptional modification is to fine‐tune ribosome–nascent peptide interactions. Structural and biochemical evidence suggest that m2A2503 may act in concert with the previously identified nascent‐peptide sensor, A2062, in the ribosome exit tunnel to relay the stalling signal to the peptidyl transferase centre.

Macrolide antibiotics allosterically predispose the ribosome for translation arrest

“…The juxtaposition of the peptide and antibiotic brings the stalling domain into contact with tunnel sensors, which relay the signal to the PTC, impairing its functions (12,14,19,27). Although such a view is sufficient to rationalize the mechanism of arrest with long regulatory peptides, it fails to explain how an antibiotic can promote arrest with the only 3-aa-long MRL peptide.…”

“…Juxtaposition of 8-9-aa long IAVV and IFVI stalling peptides with the antibiotic in the tunnel brings the peptide in contact with specific rRNA sensors in the NPET that help recognize the nascent chains and relay the arrest signal to the PTC. Mutations of such 23S rRNA residues (A2062, A2503, U1782, C2610) dramatically reduce the efficiency of stalling with ErmAL1 or ErmCL (15,19). Strikingly, neither these mutations nor changes of residues involved in recognition of other stalling peptides (G2583, U2584, U2586, A2587, U2609) (20)(21)(22) significantly affected arrest with the MRL peptide (Fig.…”

“…The PTC active site, defined as the middistance between the attacking amino group of the acceptor substrate and the carbonyl carbon atom of the donor, is marked by an asterisk. (B) The modeled position of the 9-aa-long ErmCL nascent peptide in the ribosomal tunnel obstructed by ERY (19). In the stalled complex, ErmCL is juxtaposed with the antibiotic in the tunnel.…”

“…Therefore, we introduced progressive truncations in the ermDL regulatory ORF that controls expression of the resistance methyltransferase ErmD. In the presence of ERY, translation of the wild-type 14-aa-long ErmDL peptide is arrested at the Leu (L7) codon, sandwiched between two Arg codons (R6 and R8) (18,19) (wt in Fig. 2).…”

Macrolide resistance mechanisms inEnterobacteriaceae: Focus on azithromycin

“…In the case of E. coli, the A2062G change has shown to strongly affect normal bacterial growth (Cochella & Green, 2004) while other substitutions such as C2610G, or A2503C, A2503U result in non-viability which does not support cell growth in the absence of wild-type ribosomes in E. coli (V azquez- Laslop et al, 2010Laslop et al, , 2011.…”