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Abstract
All life on earth relies on the ability to translate the genetic code into protein. DNA is transcribed first into mRNA, which is then decoded by the ribosome to produce all of the proteins in the cell. The ribosome is a large, multi-subunit entropy trap that catalyzes the assembly of each protein based on a series of codons provided by the mRNA. This reaction has been occurring for billions of years and is essential in every organism. As a result, the process is highly efficient, and extensive quality control mechanisms have evolved to maintain the fidelity of each step of protein synthesis, from initiation to termination. Ribosomes are extremely stable and the small and large subunits of the ribosome make extensive contacts with each other and with a long stretch of the mRNA. Additionally, the peptidyl transfer center maintains a water-tight seal to protect the peptidyl-tRNA from hydrolysis. The translation complex is therefore extremely stable, and must be actively disassembled after each round of translation. At the end of each protein coding reading frame, a stop codon signals the end of the peptide. Release factors make specific contacts with the stop codon and position a water molecule in the peptidyl transfer center to hydrolyze the peptidyl tRNA. Recycling factors can then disassemble the ribosome for a new round of translation. If no stop codon is present, the ribosome can not elongate or terminate the peptide. Instead, the ribosome is sequestered in a nonstop translation complex with the mRNA still bound and a peptidyl-tRNA remaining in the P site. Nonstop translation complexes arise so frequently that some estimates predict that each ribosome is bound up in a nonstop translation complex 5 times per cell division cycle. The findings in this dissertation will show that resolving nonstop translation complexes is essential in bacteria and human cells.
