Michael Pfeiffer and Dr. Allen Buskirk, Chemistry and Biochemistry

Background

Each cell within our body contains thousands of different kinds of proteins, each with a different function. Proteins provide cellular structure and perform catalysis of chemical reactions in the cell. Every protein is synthesized as ribosomes link together amino acids in the order encoded by DNA. It is therefore crucial that ribosomes function with great accuracy and proficiency to synthesize proteins quickly and correctly.

Part of the ribosome’s function is to stall the translation of an aberrant protein before it is completely synthesized. The synthesis of proteins inside a cell would cease if no rescue mechanism existed; stalled ribosomes cannot restart. In eubacteria a ribosome rescue system has evolved that frees stalled ribsosomes. This mechanism utilizes a molecule called transfer messenger RNA (tmRNA) to free the ribosome from its stalled state. One important role of tmRNA is to link a sequence of amino acids to the aberrant protein signaling it for degradation.

Ribosome stalling can occur during termination of translation. Normally, release factor proteins bind to the ribosome and cleave the bond between the growing protein and the amino acid linker (transfer RNA). Exactly how release factors perform this function is unknown. An active hypothesis is that release factor proteins cause a conformational change in the ribosome, leading to the cleavage of the protein from the ribosome by an activated water molecule. The portion of the ribosome that catalyzes this important activity is known as peptidyl hydrolase. Proteins ending in the amino acid proline often have a difficult time catalyzing termination. This is most likely due to the rigid structure of proline, which prevents a conformational change in the ribosome that consequently inhibits peptidyl hydrolase activity.

In particular, the Buskirk lab is interested in finding out more about translation termination. They have already found a mutant ribosome, C889U, which they think will shed some insight on the mechanism.

Results/Conclusions

As part of my research I was responsible for two major tasks: 1) Find more mutant ribosomes that went through normal translation termination at the sequence Ser-Pro-Stop, and 2) Perform experiments to verify that our C889U mutant ribosome does in fact bypass ribosome stalling and undergo translation termination.

Search for new mutant ribosomes

The Buskirk laboratory had previously designed a genetic selection to isolate ribosomes aberrant in stalling at the sequence Ser-Pro-Stop, a sequence pre-determined cause ribosomes stalling. Once stalling occurs, tmRNA enters the A site of the ribosome mimicking a transfer RNA (tRNA) allowing the ribosome to catalyze peptidyl transfer from the nascent peptide to the adjoining amino acylated portion of tmRNA. Once this transfer is completed, the mRNA-like portion of tmRNA encodes for a peptide sequence that eventually leads to translation termination. The Buskirk lab was able to alter the encoding region of tmRNA by cloning in the catalytic region of a toxic protein, barnase, and also insert the Ser-Pro-Stop sequence into the encoding region of the barnase protein. Thus, when ribosomes stall at the Ser-Pro-Stop sequence they are rescued by tmRNA encoding the catalytic portion of barnase which in turn results in the death of the cell. Only bacterial colonies containing mutant ribosomes that do not stall, and subsequently are not rescued by tmRNA encoding the catalytic portion of barnase, survive the selection.

Using this selection I started screening for mutant ribosomes. At first my results looked very promising; there were several colonies that were surviving the selection. However, when I sequenced the ribosomes of these colonies I found that the sequences were identical to that of the wild type ribosome. Obviously, something was wrong with the selection. Much contemplation and experimentation led to the conclusion that the selection plasmid containing the barnase protein and barnase tmRNA had somehow vanished from the competent cells used to test the selection. Therefore, I was in the process of inserting the selection plasmid into the cell line used to test the selection when my research at the Buskirk lab came to an end.

Verifying the C889U mutant

In order to verify the validity of the C889U mutant we decided to attach the same sequence that causes stalling in our selection to the end of the maltose binding protein (MBP). The tmRNA tag sequence encoded six histidine amino acids. Consequently, when stalling and rescue occurs, the protein will contain a His6 tag and be detectable through immunoblotting using an anti-His6 antibody. However, to our surprise, tagging occurred for the wild-type ribosome and, to a lesser degree, with our mutant ribosome. There appeared to be some sort of cross-reactivity of the antibody with the MBP protein which resulted in the positive Western blots for both the wild-type and mutant ribosomes.

Another way which we tested the validity of our mutant ribosome was to subject it to a different selection. Ribosome stalling also occurs at a sequence of rare arginine codons; the cell only has a limited number of arginine tRNAs that correspond to a certain codon. Consequently, if several of these codons are found in a row the cell will not have a sufficient supply of tRNAs and stalling occurs. The C889U mutant ribosome was found to bypass stalling at the Ser-Pro-Stop sequence, a mechanism that involves translation termination. Because the mechanism of stalling is different in this new system we hypothesized that our mutant ribosome would stall and subsequently be tagged with the catalytic domain of barnase causing destruction to the cell. Our hypothesis turned out to be correct. These results also confirmed that there was not a flaw or mutation in the tmRNA or the tagging mechanism which would have resulted in a bypass of stalling for our mutant.

While there are still questions left unanswered and further experiments that need to be performed, our results thus far suggest that the C889U mutation does in fact bypass ribosome stalling at the Ser-Pro-Stop sequence. Currently, the mechanism of translation termination is unknown; our mutation may provide an integral piece to this unsolved p