Effects of Trans-Acting Factors on the Translational Machinery in Yeast

Synthesis of proteins, or translation, is a complex biological process requiring the coordinated effort of numerous protein and RNA factors. Central to translation is the ribosome, a complex macromolecular complex consisting of both ribosomal RNA (rRNA) and ribosomal protein (r-protein). Ribosomes are essential and are one of the oldest and most abundant biomolecules across all forms of life. In addition to the ribosome, translation requires messenger RNA (mRNA), transfer-RNA conjugated to an amino acid (aa-tRNA), translation factors, and energy in the form of ATP and GTP. Translation universally occurs in four major stages, initiation, elongation, termination, and recycling, with initiation serving as the key regulatory and rate-limiting step. The goal of this thesis was to investigate key factors regulating this step. First, a cell-free translation system from the budding yeast Saccharomyces cerevisiae was developed, allowing fine-tuned control of mRNA elements such as the 5’ cap structure. This method was used to uncover a previously undescribed cap-independent translation enhancer from the Black Beetle Virus. Secondly, this method was developed further to utilize purified ribosomes to assay the effect of chemical stress on the ribosome and translation efficiency. This method is a powerful alternative to in vivo approaches since ribosomes can be selectively damaged and assayed without risking contamination of other key molecules like tRNA, mRNA, and translation factors