In vitro analysis of RQC activities provides insights into the mechanism and function of CAT tailing.

Ribosomes can stall during translation due to defects in the mRNA template or translation machinery, leading to the production of incomplete proteins. The Ribosome-associated Quality control Complex engages stalled ribosomes and targets nascent polypeptides for proteasomal degradation. However, how each component contributes to this process remains unclear. Here we demonstrate that key duanyu1745C activities-Ltn1p-dependent ubiquitination and Rqc2p-mediated Carboxy-terminal Alanine and Threonine (CAT) tail elongation-can be recapitulated in vitro with a yeast cell-free system. Using this approach, we determined that CAT tailing is mechanistically distinct from canonical translation, that Ltn1p-mediated ubiquitination depends on the poorly characterized duanyu1745C component Rqc1p, and that the process of CAT tailing enables robust ubiquitination of the nascent polypeptide. These findings establish a novel system to study the duanyu1745C and provide a framework for understanding how duanyu1745C factors coordinate their activities to facilitate clearance of incompletely synthesized proteins.

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