Novel strategies to explore the mechanism of protein synthesis at the single-molecule scale

Enzymes are inherently dynamic macromolecules that operate on a range of timescales. Identifying and understanding key conformational dynamics of enzymes that are directly related to their catalytic function remains of significant importance. The process of translation is catalyzed by one such highly dynamic enzyme known as the ribosome. The aim of this dissertation is to understand this process at the single-molecule scale using Fluorescence Resonance Energy Transfer (FRET), which can help relate how physical snapshots of the ribosome structure to their function.;The translation process is characterized by repetitive, messenger RNA (mRNA) codon-dependent binding of aminoacyl-tRNA (aa-tRNA) at the A site of the ribosome (tRNA selection) punctuated by large-scale movements of tRNA with respect to the ribosome (translocation). To accurately transmit information found in the mRNA into a corresponding polypeptide, both elemental translation reaction must occur with high fidelity. To investigate the role of dynamic processes in the ribosome and tRNA occurring during aa-tRNA selection and translocation single-molecule FRET techniques have been developed. Critical to this platform for biophysical study is the development of novel strategies for site-specific introduction of fluorophores at key positions within the ribosome that report on time-dependent conformational changes relevant to function. It is also imperative to develop novel approaches for long-lasting and photostable fluorophores in order to study the process of protein synthesis which occurs on the order of several minutes.;Chapter 1 and 2 focus on the current state of knowledge on bacterial elongation and the preparation of samples and methods used to study this process using smFRET. Chapter 3 is an investigation aimed at exploring mechanisms to minimize photophysical phenomena inherent to fluorophores. Such methods are necessary to avoid interpreting biologically irrelevant signals as discrete, dynamic tRNA selection steps. Small-molecule additives, in particular additives that serve as triplet state quenchers, have been used to significantly reduce fluorophore artifacts and improve their photostability. Chapter 4 explores the aa-tRNA selection process using multiple structural perspectives, where dynamics are thought to play a key role in the fidelity mechanism. Fluorescently labeled P-site tRNA and ribosomal protein L11 were used to determine that the reversible motions of the incoming tRNA play a central role in the selection process and the fidelity of peptide synthesis. Chapter 5 seeks to understand the role of the L12 stalk during tRNA selection through multiple structural perspectives . Fluorescently-labeled L12, L11, EF-Tu and aa-tRNA were used to demonstrate that a single CTD of L12 assists in the initial binding of the ternary complex to the ribosome, directly facilitating EF-Tu's interaction with GAC to stabilize the state where GTP hydrolysis can occur. Finally the dissertation concludes with Chapter 6 which draws conclusions from this dissertation and presents possible future directions this work might take. This includes watching processive translation using smFRET which can help detect conformational intermediates involved in translation regulation.