Diana Valverde and Dr. Allen Buskirk, Chemistry and Biochemistry
The ribosome is a macromolecular machine found in all living cells. It catalyzes protein synthesis through a process known as translation. The ribosome reads messenger RNA that carries the blueprint for a protein product and brings in the appropriate amino acids to build a specific protein. Ribosomes are composed of two subunits (50S and 30S) that form three binding sites for transfer RNA. The A site of the ribosome accepts the incoming tRNA charged with an amino acid. This tRNA transfers its amino acid to a growing polypeptide chain held in the P site. After the transfer, the tRNA exits the ribosome through the E site. Previous studies revealed that certain nascent–newly forming–peptide chains stall ribosomes during protein synthesis. Stalling has been shown to play a significant role in gene regulation. For example, SecM is a peptide that stalls the ribosome when there are low levels of the bacterial-motor protein SecA. This stalling event allows the ribosome to synthesize more SecA protein. Previous studies have shown that only a few amino acids are required for stalling. These known stalling peptide sequences are short and simple but lack similarity and show tolerance to mutation. This suggests that there may be undiscovered stalling motifs within other E. coli protein. We used a new genetic selection, called the two-hybrid system, to find additional stalling sequences from a collection of random sequences (a library). This library consisted of 108 sequences, each having twenty random amino acids. We found several novel stalling sequences, and my responsibility was to locate the exact site of stalling in the ribosome. I performed deletions to isolate the minimal sequence motif needed for stalling and performed mutagenesis studies to find which amino acids are critical for stalling the ribosome. From this data, we expected to formulate rules as to what is necessary and sufficient for stalling. These rules we could then be used in the future to identify endogenous proteins where stalling may play a role in gene expression or protein folding.
The first step is to perform a toe-print assay on the full peptide stalling sequence to determine if and where the ribosome stalls. In toe-printing, we use the PURE Express in vitro transcription/translation kit. This kit converts DNA containing stalling sequences into mRNA which is translated by the ribosome. Ribosomes stop on the stalling sequence in the mRNA, at which point reverse transcriptase (RT) and radiolabeled NV1 primer are added to the reaction mixture. RT copies the mRNA to DNA until it encounters the ribosome and stops. These DNA fragments are run on denaturing polyacrylamide gel electrophoresis (PAGE) and visualized with a phosphoimager. Bands corresponding to the location of the ribosome on the mRNA are easily located, from which the exact stalling site will be identified. We will repeat the same process with each stalling sequence. Since it is not feasible to mutate every amino acid to determine the exact stalling motif, we are going to progressively delete groups of four amino acids from the Nterminus of the selected 20-mers. We will perform toeprinting on the deletion constructs. If deletion of certain residues reduces the toeprint intensity, then these residues are necessary for stalling. We will then mutate each amino acid in this region individually to alanine. These experiments will identify which residues are necessary and sufficient for stalling. Based on our findings, we can look for patterns in the peptide sequences to come up with a set of guidelines that predict stalling motifs in endogenous proteins. These endogenous proteins can further be studied to identify the role of stalling, whether it is for gene regulation or protein folding.
We were successful in identifying the exact site of stalling. Unfortunately, after progressively deleting amino acids in groups of four from the N-terminus of the selected 20-mers, there was no significant decrease in the intensity of toe-print bands. We decided this wasn’t a robust way to find the amino acids that were significant for stalling. Additionally, we found this would require more work and time to obtain results, prolonging our research. Fortunately, we were able to design immediately an alternative experiment to continue our research investigation.
We decided to perform mutagenesis and reselection of those stalling sequences we had previously found. We did this through the process of polymerase chain reaction (PCR), where a degenerate 3’-primer causes each base encoding the twenty amino acid stalling motif to be mutated at 30%. This created a mini library containing samples with sequences very similar to the original stalling sequence. This library is then subject to our two-hybrid selection and hundreds of surviving clones are sequenced. Essential residues are conserved whereas nonessential residues are free to vary. A weblogo was used to show the alignments of active mutants that are conserved. One of our sequences, numbered 65, revealed that DTS (aspartic acidthreonine- serine) are essential for stalling. Further analysis of nucleotide alignment showed that the third base of each codon in the DTS sequence varied freely, while the first two bases are conserved. This suggests that stalling may be caused by the peptide and not the RNA sequence.
The next chapter of our research investigation is to show how translation rates are affected by stalling peptides. The data we obtained showed that very short peptide sequences cause stalling in vitro and in vivo. This raises the question: why would a protein inhibit its own translation? We hypothesize that stalling peptides may be capable of fine-tuning the rate of translation in order to control protein folding. As a first step, we will measure the rate of synthesis of a protein containing short stalling motifs in vivo.
Research has been a rewarding experience for me in the sense that I’ve had to overcome my feelings of frustration when things didn’t work and instead try to find other means to make my experiments work. Through my personal investigator, Dr. Allen Buskirk, and mentor, Christopher Woolstenhulme, I have learned how to approach these situations by observing their methodology of dealing with and coming up with new ideas to make our experiments work. I have learned to develop patience, remembered that science is not only about work but having. Our work is still in progress and I continue to work with Dr. Buskirk’s lab. Much progress has been made and we anticipate our research will be published in a scientific journal by the summer of 2012. I have already started the next phase of our research investigation. We did a blast search and found several E.coli proteins that contain one of the novel amino acid stalling sequences (PPP) we found in our genetic selection. My role this year is to perform mutagenesis on two of these several stalling E. coli proteins, LigT and LepA, to study the effect of translation rates. Both of these proteins exhibit the amino acid sequence PPP, where the 2nd proline stalls in the Psite. Studying their translation rates will not only provide further evidence for stalling, but also make our research applicable to natural organism systems. We expect the translation rates of to exhibit LigT and LepA proteins to be slower than non-stalling peptide sequences.