Chapter 17 Studying tmRNA‐Mediated Surveillance and Nonstop mRNA Decay

Abstract

In bacteria, ribosomes stalled at the 3'-end of nonstop or defective mRNAs are rescued by the action of a specialized ribonucleoprotein complex composed of tmRNA and SmpB protein in a process known as trans-translation; for recent reviews see Dulebohn et al. [2007], Keiler [2007], and Moore and Sauer [2007]. tmRNA is a bifunctional RNA that acts as both a tRNA and an mRNA. SmpB-bound tmRNA is charged with alanine by alanyl-tRNA synthetase and recognized by EF-Tu (GTP). The quaternary complex of tmRNA-SmpB-EF-Tu and GTP recognizes stalled ribosomes and transfers the nascent polypeptide to the tRNA-like domain of tmRNA. A specialized reading frame within tmRNA is then engaged as a surrogate mRNA to append a 10 amino acid (ANDENYALAA) tag to the C-terminus of the nascent polypeptide. A stop codon at the end of the tmRNA reading frame then facilitates normal termination and recycling of the translation machinery. Through this surveillance mechanism, stalled ribosomes are rescued, and nascent polypeptides bearing the C-terminal tmRNA-tag are directed for proteolysis. Several proteases (ClpXP, ClpAP, Lon, FtsH, and Tsp) are known to be involved in the degradation of tmRNA-tagged proteins (Choy et al., 2007; Farrell et al., 2005; Gottesman et al., 1998; Herman et al., 1998, 2003; Keiler et al., 1996). In addition to its ribosome rescue and peptide tagging activities, trans-translation also facilitates the selective decay of nonstop mRNAs in a process that is dependent on the activities of SmpB protein, tmRNA, and the 3' to 5'-exonuclease, RNase R (Mehta et al., 2006; Richards et al., 2006; Yamamoto et al., 2003). Here, we describe methods and strategies for the purification of tmRNA, SmpB, Lon, and RNase R from Escherichia coli that are likely to be applicable to other bacterial species. Protocols for the purification of the Clp proteases, Tsp, and FtsH, as well as EF-Tu and other essential E. coli translation factors may be found elsewhere (Joshi et al., 2003; Kihara et al., 1996; Makino et al., 1999; Maurizi et al., 1990; Shotland et al., 2000). In addition, we present biochemical and genetic assays to study the various aspects of the trans-translation mechanism.