Investigating the properties of translational arrest motifs: interactions between the nascent chain and the ribosome

[摘要] Ribosomes are responsible for the synthesis of all cellular proteins.It was initially believed that translating nascent chains would not interact with the ribosome exit tunnel, however, a small but increasing number of proteins have been identified that interact with the exit tunnel to induce translational arrest.Escherichia coli (E.coli) secretion monitor (SecM) is one such stalling peptide.SecM monitors the SecYEG translocon export activity through its own translocation to the periplasm and upregulates translation of SecA, an ATPase involved in the SecYEG translocation machinery, when translocation is reduced.How stalling peptides interact with the ribosome exit tunnel is not fully understood, however, a key feature required is an essential amino acid arrest motif at their C-terminus, and additionally some peptides, including SecM, undergo compaction of the nascent chain within the exit tunnel upon stalling.In this study analysis of SecM peptides with both alanine and conservative mutations of key arrest motif residues were investigated.This identified three conservative mutants that can retain a degree of stalling; and this level of stalling is further increased when coupled with mutation of a non-essential arrest motif residue P153A.Further analysis of these mutants by pegylation assays indicates that this increase in stalling ability is due to the ability of the P153A mutation to reintroduce compaction of the nascent chain within the exit tunnel, possibly due to the improved flexibility of the nascent chain provided by the removal of the restrictive proline residue. These methods highlight the significance of the interactions between the nascent chain and the exit tunnel, which contribute to translation arrest.This study also examines the ability of stalling peptides to undergo translation arrest in ribosomes of alternative domains, investigating in particular the ability of E.coli SecM and TnaC and fungal Neurospora crassa (N.crassa) AAP to arrest in eukaryotic Wheat Germ and prokaryotic E.coli ribosomes.This study concludes that stalling peptides only induce translation arrest in ribosomes of the same domain.In addition, it also revealed the ability of inducible stalling peptides to undergo translational pausing, prior to the commitment to full translation arrest, a process that does not appear to occur in intrinsic stalling peptides.

[发布机构] University:University of Glasgow;Department:Institute of Molecular Cell and Systems Biology

[关键词] Ribosome, translation, stalling, arrest, nascent chain, compaction, SecM, TnaC, AAP, pegylation