Browsing by Author "Lin, Ran"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Amino Acid purification and Single-Molecule FRET study of tRNA translocation(2019) Kapadia, Taher; Lin, RanUnderstanding the basic translational mechanisms of the ribosome during protein translation is essential to developing novel chemical defenses against the increasing amount of anti-biotic resistant bacteria. To study the fundamentals of the protein building process, we can employ smFRET studies which require debris-free, pure samples of amino acids, the building blocks of all proteins. Our experiment focused on the overexpression and purification of the Glutamic Acid tRNA and its translocation in the ribosome. This was accomplished by inserting a Glutamic Acid sequence into a vector plasmid through a series of digestion and ligation reactions. The recombinant plasmid was then overexpressed in E. Coli and the tRNA was purified through chloroform extraction, gel filtration, and ion-exchange chromatography. The pure sample was used to make a ribosome complex and the translocation of Glutamic Acid from the A site in the ribosome to the P site was studied through smFRET. From the results, it was concluded that the ribosome can efficiently process the Glutamic Acid tRNA when a frameshifting motif is not present. This raises the question of what may occur if frameshifting motifs are added into an mRNA transcript and how this may affect the trans-locational process of glutamic acid. The future of this study should target the translational process in the presence of frameshifting motifs to identify targets which can halt the building of the proteins. Studies attempting to apply such mechanisms to cells in a host should, however, be mindful that both the bacterial and host ribosomes are susceptible to frameshifting which may limit or complicate discovery of target sites.Item The Mechanism of Ribosomal Translocation Revealed by Single-molecule FRET and Super-resolution Force Spectroscopy (SURFS)(2020-12) Lin, Ran; Wang, Yuhong; Fox, George E.; Fujita, Masaya; Cai, ChengzhiDuring protein synthesis, peptidyl tRNA at the A site moves to the P site, and deacylated tRNA at the P site moves to the E site. This step in protein translation is called translocation and is catalyzed by a conserved GTPase--elongation factor G (EF-G). This dissertation addresses the role of EF-G and EF-G-induced GTP hydrolysis in the typical translocation event. In addition, we evaluate the role of EF-G in abnormal translocation events like ribosomal stalling and frameshifting during the translation process. The rate of tRNA translocation during protein translation is accelerated 50-fold by the EF-G-induced GTP hydrolysis. It is not clear that the GTP hydrolysis causes some conformational changes in EF-G or powers tRNAs translocation directly. To achieve our experimental objectives, we developed a dual FRET system to track the internal interaction of two EF-G domains while simultaneously tracking the interaction of ribosomes and tRNAs. The FRET data shows that GTP hydrolysis and ribosome are required for EF-G conformational change but are not coupled directly with tRNA translocation. Meanwhile, translocation can be paused when the ribosome stalls on an mRNA encoding a polyproline sequence. One possible reason for ribosomal stalling is the slow peptide bond formation between prolines. The FRET system was used to track sensitive changes in the interaction of tRNAs and ribosomes to detect the kinetics of the prolyl peptide bond formation. Our observations suggest that ribosomal stalling is caused by the destabilization of A-site tRNAs in the cases where several consecutive proline codons enter the mRNA decoding center. Furthermore, translocation can also be misled by ribosomal frameshifting. This process is influenced by both the mRNA sequence and secondary mRNA structure. Super-resolution force spectroscopy (SURFS) was used to determine the force in the process of ribosomal translocation precisely. Neomycin was applied to trap different phases of ribosomal frameshifting. Surprisingly, four stages were observed when studying the movements at the 5'-end of mRNA by SURFS. It was found that during mRNA translation, its 5' terminus is always more motile than the 3' terminus. It was concluded that EF-G rescues the frameshifting by stabilizing the ribosome with compromised conformational changes.