Abstract
Alternative ribosome-rescue factor B (ArfB) rescues ribosomes stalled on non-stop mRNAs by releasing the nascent polypeptide from the peptidyl-tRNA. By rapid kinetics we show that ArfB selects ribosomes stalled on short truncated mRNAs, rather than on longer mRNAs mimicking pausing on rare codon clusters. In combination with cryo-electron microscopy we dissect the multistep rescue pathway of ArfB, which first binds to ribosomes very rapidly regardless of the mRNA length. The selectivity for shorter mRNAs arises from the subsequent slow engagement step, as it requires longer mRNA to shift to enable ArfB binding. Engagement results in specific interactions of the ArfB C-terminal domain with the mRNA entry channel, which activates peptidyl-tRNA hydrolysis by the N-terminal domain. These data reveal how protein dynamics translate into specificity of substrate recognition and provide insights into the action of a putative rescue factor in mitochondria.
Highlights
Alternative ribosome-rescue factor B (ArfB) rescues ribosomes stalled on non-stop mRNAs by releasing the nascent polypeptide from the peptidyl-tRNA
We first used kinetic experiments in a fully reconstituted in vitro system to study whether the activity of ArfB depends on the length of the mRNA in the stalled ribosome complexes
When more than 9 nt of the mRNA extended past the P-site codon, the rate of hydrolysis reaction decreases sharply, by about 15-fold (P + 12) to 100-fold (P + 99) (Fig. 1c), indicating that ArfB-mediated rescue is most efficient when the mRNA is short
Summary
Alternative ribosome-rescue factor B (ArfB) rescues ribosomes stalled on non-stop mRNAs by releasing the nascent polypeptide from the peptidyl-tRNA. Engagement results in specific interactions of the ArfB C-terminal domain with the mRNA entry channel, which activates peptidyl-tRNA hydrolysis by the N-terminal domain. These data reveal how protein dynamics translate into specificity of substrate recognition and provide insights into the action of a putative rescue factor in mitochondria. A comprehensive mutagenesis study identified ArfB residues that are important for ribosome binding and peptidyl-tRNA hydrolysis[10]. Most of these essential amino acids are not located in the catalytic N-terminal domain, but in the C-
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