Abstract:

Bacteria regulate protein synthesis during environmental stress as a survival mechanism. One way translation is regulated is through cleavage of ribosome-bound mRNA by ribosome-dependent toxins. This mRNA cleavage stops the synthesis of the protein encoded by the cleaved-mRNA, conserves nutrients and likely plays an important, yet unknown, role in altering the spectrum of proteins translated during stress. A very unique feature of ribosome-dependent toxins is that they can recognize and cleave several mRNA sequences on the ribosome. In this dissertation, the molecular mechanism of recognition and cleavage of adenosine-rich mRNA codons by the Proteus vulgaris HigB toxin, which was originally identified on a drug-resistance plasmid from a P. vulgaris urinary tract infection, was investigated. Structural and biochemical studies reveal that the HigB toxin displays degenerate substrate specificity by creating two A-site nucleotide-binding pockets capable of interacting with numerous nucleotides. Surprisingly, the third nucleotide-binding pocket of HigB is adenosine-specific. Recognition of the third A-site nucleotide appears to be a distinct feature of ribosome-dependent toxins and likely influences which mRNAs are targeted for cleavage during environmental stress.<br/><br/>Ribosome-dependent toxins must be highly regulated. The HigA antitoxin binds to and inactivates the HigB toxin when cells are not in a stressed state. Structural investigation shows that HigA and HigB form a tetrameric complex consisting of two HigA proteins and two HigB proteins. This structure reveals that HigA does not inactivate HigB through direct interactions with the HigB active site, as observed in many other toxin-antitoxin complexes. Instead, HigA binding to HigB likely inhibits HigB by blocking association of HigB with the ribosome. The knowledge of how HigB activity is regulated and its unique specificity provides a molecular framework for scientists to uncover how ribosome-dependent toxins control translation during environmental stress.