Adaptor Guided Proteolysis Initiates on the Ribosome

Objective To characterize the capture of tmRNA‐tagged proteins by the SspB adaptor on the ribosomes. Methods We utilized in vitro enzyme kinetics and in vivo ribosome enrichment assays to study SspB‐ribosome interactions and the consequential degradation of tagged protein. We isolate total ribosomes and determine the relative amounts of SspB associated with the ribosomes by specifically enriching for ribosomes translating the reporter mRNA of choice. Results Adaptor proteins extend the substrate range and specificity of AAA+ proteases. The SspB adaptor protein enhances the efficiency of ClpXP mediated proteolysis of tmRNA‐tagged proteins. Our investigations into whether ribosomes serve as a hub for initiating directed proteolysis reveal that SspB captures tag‐bearing proteins on tmRNA‐recued ribosomes in a trans ‐translation dependent manner, in a process reliant on distinct recognition signals within the tmRNA degron. Defined alterations in the SspB and ClpX recognition elements of the tmRNA peptide sequence, or the peptide‐binding groove of SspB, have adverse effects on the efficiency of SspB ribosome recruitment and degradation of marked proteins. Conclusions Our studies suggest that SspB scans translating ribosomes to capture tmRNA tagged proteins and guide their delivery to ClpXP for directed proteolysis. Our inquiries have also unveiled the capacity of SspB to capture additional nascent polypeptides that harbor genetically encoded yet degenerate forms of the tmRNA degron and modulate their intracellular abundance through selective proteolysis. Support or Funding Information NIH