Jeremy Goodson and Dr. Allen Buskirk, Chemistry and Biochemistry

Trans-translation can be broken down into 5 steps. , After a ribosome stalls, the mRNA in its aminoacyl-tRNA site (A-site) is cleaved and released, which leaves the A-site empty. The key molecule in ribosomal rescue, transfer-messenger RNA (tmRNA), then forms a complex with Small Protein B (SmpB). This complex is recruited into the empty A-site of the stalled ribosome. tmRNA contains a message in an open reading frame, which encodes a degradation tag that will be added to the protein later. In the next surprising step, the ribosome switches RNA templates from the stalled mRNA to tmRNA, resuming translation on the mRNA portion of tmRNA and synthesizing a degradation tag (amino acid sequence: AANDENYALAA) at the end of the protein. The ribosome reaches a stop signal in the tmRNA open reading frame, the tagged protein is released and degraded by proteases, and the ribosome is rescued.

Our lab was interested in identifying the endonuclease that cleaves mRNA in stalled ribosomes. To investigate, we created a series of mutant E. coli strains, each bearing ribosomes with only a single mutation. We identified three mutations that inhibit tmRNA function and tagging: A1152 deletion, T1123 deletion, and A1152G (fig. 1). We identified these mutants with an assay called the barnase assay that allows cells to survive only if they cannot perform trans-translation. In order to confirm that these mutants prevent trans-translation, we performed an immunoblot.
An immunoblot uses an antibody to detect the presence of a protein. Because antibodies have highly specific binding properties, only the protein of interest, MBP in this case, will bind to the antibody. Trans-translation (tagging) results in a detectable, tagged protein on an immunoblot. We developed a special tmRNA, which encodes a detection signal in place of the degradation tag. I then prepared a plasmid containing the MBP gene. I prepared these plasmids with restriction enzymes (enzymes that cut DNA at a known nucleotide sequence) and PCR (polymerase chain reaction, used to make additional copies of DNA). After cutting an existing plasmid open with restriction enzymes, I inserted the MBP gene into the plasmid. The MBP gene contained a proline-stop sequence, which induces ribosome stalling, trans-translation and tagging. Therefore, after MBP is synthesized, it will not be tagged if the mutant ribosomes prevent trans-translation. Because the tag is detectable in an immunoblot, we can distinguish between tagged and untagged protein and thereby determine whether or not the mutant ribosomes prevent trans-translation. The immunoblot confirmed that MBP was synthesized (figure 2), but that it was not tagged (figure 3).

If in subsequent testing we find that the mutant ribosomes prevent trans-translation, then we will test whether or not they affect the endonucleolytic cleavage of A-site mRNA, by preparing a plasmid, which codes for two types of mRNA. The mutant ribosome will stall in the middle of type I (mRNA in the A-site) and at the end of type II (empty A-site). Because type II mRNA will cause the ribosome to stall with an empty A-site (the same as if the endonuclease had just cut the A-site mRNA), we expect mutant ribosomes that inhibit step A of trans-translation to cause tagging only in the type II mRNA.

References

• Hayes, C. S.; Sauer, R. T. Cleavage of the A Site mRNA Codon during Ribosome Pausing Provides a Mechanism for Translational Quality Control. Molecular Cell 2003, 12, 903-911.
• Withey, J. H.; Friedman, D. I. A Salvage Pathway For Protein Synthesis: tmRNA and Trans-Translation. Annu. Rev. Microbiol. 2003, 57, 101-1