Abstract
Ribosomes are responsible for the synthesis of all cellular proteins. Due to the diversity of sequence and properties, it was initially believed that translating nascent chains would travel unhindered through 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. How and why these peptides interact with the exit tunnel is not fully understood, however key features required for stalling appear to be an essential peptide arrest motif at the C-terminus and compaction of the nascent chain within the exit tunnel upon stalling. Mutagenesis of the SecM arrest sequence has identified three conservative point mutations that can retain a degree of stalling in this highly conserved sequence. 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 activity during translation 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 a restrictive proline residue. The data presented here suggest that arrest sequences may be more prevalent and less highly conserved than previously thought, and highlight the significance of the interactions between the nascent chain and the exit tunnel to affecting translation arrest.
Highlights
For ribosomes to translate thousands of peptides efficiently, non-specific interactions with the ribosome tunnel must be avoided, to prevent slowing the rate of translation
Arg163 is a key residue in the Secretion monitor (SecM) peptide and cannot tolerate alteration, even a conservative mutation to lysine results in loss of the SecM stalling ability
Interaction between arginine and the ribosome exit tunnel does not appear to be transient as it is highly specific. Instead it suggests that the arginine residue may bind within a specific rRNA pocket and interact with rRNA nucleotide A2062, to induce structural changes that signal to peptidyl transferase centre (PTC), leading to the ratcheting of the tRNA moiety and translational stalling [14]
Summary
For ribosomes to translate thousands of peptides efficiently, non-specific interactions with the ribosome tunnel must be avoided, to prevent slowing the rate of translation. This suggests that the residues further away from the C-terminus, while not forming essential interactions with the c 2019 The Author(s) It remained to be explored whether increased freedom of movement out with the essential arrest motif, through mutation of non-essential Pro146 to alanine could influence positioning of the key residues at the C-terminus of the SecM nascent chain. These results illustrate the significance of each individual arrest motif residue and their interactions with the exit tunnel that contribute to translation arrest
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