David Healey and Dr. Allen Buskirk, Chemistry and Biochemistry

During the last year, I have worked in collaboration with Allen Buskirk and DeAnna Cazier to study the interactions between SmpB and tmRNA during a process called bacterial trans-translation. To understand our experiments, it is necessary to know a little about what trans-translation is, and how it works.

All living things need proteins to live and grow. Organisms make proteins with very complex molecular machines called ribosomes. In the process called translation, ribosomes “read” long molecules called mRNA, and link together amino acids in the appropriate order to create long and complex proteins. Occasionally, ribosomes stall and can no longer complete the chain. If the ribosomes remain like that, the cell will die. Bacteria have evolved an interesting mechanism to rescue their stalled ribosomes using two small molecules called tmRNA and SmpB. tmRNA enters the ribosome and displaces the stuck mRNA. Then somehow the ribosome “jumps” from the mRNA template to the tmRNA itself, and finishes translating normally. The ribosome is free to continue making proteins. Because the ribosome has jumped from one template to another, the process is called ¬trans-translation.

Why study trans-translation? Trans-translation is important because a large number of current antibiotics specifically target bacterial ribosomes, causing them to stall. Trans-translation makes bacteria less susceptible to antibiotics. Furthermore, understanding the molecular processes involved with ribosomes may lead to future advances in antibiotic research.

It is already well established that SmpB is necessary for proper trans-translation, but the hypothesis we set out to test was whether SmpB helps position the tmRNA template correctly within the ribosome, and, if it does, identify which parts of SmpB are necessary for the correct frame to be set.

Initially we did a full random mutation of SmpB using error-prone PCR, which generated around ten million different random SmpB mutants. We then ran them through a selection whereby a kanamycin-resistance protein will only be formed properly if trans-translation begins shifted one nucleotide early. In other words, we were looking for an SmpB mutant that affects trans-translation by altering where the ribosome resumes translating on tmRNA.

Out of the ten million mutants, we found three that survived the selection. We dubbed them A1, A2, and A5, and sequenced all three. The A1 clone has two changes, Tyr24Cys and Val129Ala. A2 has these and the additional Glu107Val mutation. A5 shares the same Tyr24Cys mutation as A1 and A2 but coupled instead with Ala130Gly.

The next thing we did was verify that A1, A2, and A5 really affected the frame during trans-translation. We did this with a process called immunoblot. We engineered a protein, GST, to cause stalling. Then, when the ribosome is rescued by tmRNA, the tmRNA adds a tag that can be recognized and bound by fluorescent antibodies, but only if the tag is added in the correct frame. So with a fluorescent imager we were able to compare the tagging activity of cells with normal SmpB with the tagging activity of cells containing the SmpB mutants A1, A2, and A5. We found that, in particular, the A5 mutant consistently changed the frame selection, but on an already-mutate tmRNA, and not on wild type tmRNA (p<0.01). What this means is that the mutations in SmpB are not enough alone to alter the reading frame, only that they do have that affect on that particular mutant tmRNA.

When mapped onto the atomic structure of Thermus thermophilus SmpB,17 the three SmpB residues that are changed in the A1, A2, and A5 mutants cluster in a junction at the bottom of the protein, at the opposite end from the well-characterized tmRNA binding site. The clustering of these mutations in this single site implicates this junction at the bottom of SmpB in the frame selection process.

The mutant tmRNA that we had used for the selection was one which we previously found to causes the frame to shift up one position nearly every time. It is called A86C because it has a cytosine (C) instead of an adenine (A) at position 86, right before the start site on the tmRNA. We showed that position to be important to the frame selecting process earlier, and had hypothesized that SmpB perhaps bound that spot in order to position the tmRNA correctly in the ribosome. Because the mutant SmpBs only rescue the affects of A86C, we conclude that the mutants broaden the specificity of the protein to work with both A and C instead of just C.

Those two findings together help establish our assertion that SmpB does affect the reading frame of ribosomes during trans-translation. If the mutants had worked on normal, wild-type tmRNA, we might have more strongly concluded that those specific amino acids affect where the ribosome chooses to resume translation on tmRNA, which would have been a good indication of a place on SmpB that actually binds tmRNA to position it within the ribosome. But because the effect was limited to the A86C mutant, we had to soften our conclusion to just pointing out that we had found an instance of SmpB affecting the reading frame, which, in and of itself was enough to merit publication in the Journal of Molecular Biology.