Critical 23S rRNA interactions for macrolide-dependent ribosome stalling on the ErmCL nascent peptide chain

Abstract

The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and co-translational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood. Here, we used a nucleotide analog interference approach to unravel important functional groups on 23S rRNA residues in the ribosomal exit tunnel for ribosome stalling on the ErmC leader peptide. By replacing single nucleobase functional groups or even single atoms we were able to demonstrate the importance of A2062, A2503 and U2586 for drug-dependent ribosome stalling. Our data show that the universally conserved A2062 and A2503 are capable of forming a non-Watson–Crick base pair that is critical for sensing and transmitting the stalling signal from the exit tunnel back to the peptidyl transferase center of the ribosome. The nucleobases of A2062, A2503 as well as U2586 do not contribute significantly to the overall mechanism of protein biosynthesis, yet their elaborate role for co-translational monitoring of nascent peptide chains inside the exit tunnel can explain their evolutionary conservation.

In vitro ribosome synthesis and evolution through ribosome display

“…Of the CR mutants that were viable in vivo, there was no clear relationship between fold enrichment after RISE and cellular growth rate. Interestingly, the non-viable CR variants all contained mutations at the 2062 position, which is consistent with the previous observation that such mutations are lethal despite in vitro ribosome activity 32,35 . The result that ribosomes that are active in iSAT may be unable to support life demonstrates the potential of RISE to select ribosomes that are proficient at translating challenging templates or monomers in vitro without concern for viability in living cells.…”

In vitro ribosome synthesis and evolution through ribosome display

“…Of the CR mutants that were viable in vivo, there was no clear relationship between fold-enrichment after RISE and cellular growth rate. Interestingly, the non-viable variants all contained mutations at the 2062 position, which is consistent with the previous observation that such mutations are lethal despite in vitro ribosome activity 20,23 . The result that ribosomes that are highly active in vitro may be inviable in vivo demonstrates the potential of RISE for selecting ribosomes that are proficient at translating challenging templates or monomers without concern for in vivo viability.…”

The Ribosomal Protein uL22 Modulates the Shape of the Protein Exit Tunnel

“…Biochemical, genetic, and structural studies have shown that the tunnel interacts with specific sequence motifs of nascent chains. These interactions may lead to translation arrest that regulates gene expression (Arenz et al, 2014;Bischoff et al, 2014;Chiba et al, 2011;Gong and Yanofsky, 2003;Koch et al, 2017;Nakatogawa and Ito, 2002;Ramu et al, 2011;Seidelt et al, 2009;Sohmen et al, 2015;Vazquez-Laslop et al, 2011;Woolhead et al, 2006).…”