Abstract

Ribosomes stall on defective messenger RNA transcripts in eubacteria. Without a mechanism to release stalled ribosomes, these cells would die. Transfer-messenger RNA (tmRNA) and small protein B (SmpB) reactivate stalled ribosomes in a process known as trans-translation. Together, tmRNA and SmpB mimic alanyl-tRNA, entering the A site of stalled ribosomes and accepting transfer of the stalled polypeptide. A portion of tmRNA is then positioned as a template for the ribosome to resume translating. The tmRNA open reading frame encodes a proteolysis tag to mark the aberrant polypeptide for degradation and a stop codon to release the ribosome. How are tmRNA and SmpB allowed into stalled ribosomes? In normal translation, decoding mechanisms carefully monitor the anticodon of tRNAs entering the A site and select only those that are complementary to the mRNA codon. How do tmRNA and SmpB get around the decoding machinery? It appears that interactions between the SmpB C-terminal tail and the decoding center are responsible. Using an in vivo tagging assay and an in vitro peptidyl-transfer assay, we monitored the effect of mutations in the SmpB tail on trans-translation. We found that mutations in SmpB that prevent helix formation are unable to support peptidyl transfer. We also found that while mutation of key nucleotides in the ribosomal decoding center severely inhibit peptidyl transfer to normal tRNAs, these mutations do not inhibit peptidyl transfer to tmRNA. We conclude that the SmpB tail stimulates peptidyl transfer by forming a helix that interacts with the ribosome to signal decoding in a novel manner. How is the tmRNA open reading frame positioned for the ribosome to resume translating? Mutation of the tmRNA nucleotide A86 alters reading frame selection. Using a genetic selection, we identified SmpB mutants that restore normal frame selection to A86C tmRNA without altering frame selection on wild-type tmRNA. Through rational mutation of the SmpB tail we identified an SmpB mutant that supports peptidyl transfer but prevents translation of the tmRNA open reading frame. We conclude that SmpB plays a functional role in selecting the tmRNA open reading frame.

Degree

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

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