Single-Molecule Analysis of Ribosome and Initiation Factor Dynamics during the Late Stages of Translation Initiation Daniel David MacDougall Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2012 ©2012 Daniel David MacDougall All Rights Reserved Abstract Single-Molecule Analysis of Ribosome and Initiation Factor Dynamics during the Late Stages of Translation Initiation Daniel David MacDougall Protein synthesis in all organisms is catalyzed by a highly-conserved ribonucleoprotein macromolecular machine known as the ribosome. Prior to each round of protein synthesis in the cell, a functional ribosomal complex is assembled from its component parts at the start site of a messenger RNA (mRNA) template during the process of translation initiation. In bacteria, rapid and high-fidelity translation initiation is promoted by three canonical initiation factors: IF1, IF2, and IF3. In this thesis, I report the use of single-molecule fluorescence methods to study the role of the initiation factors and ribosome-factor interactions in regulating molecular events that occur during late stages of the translation initiation pathway. In Chapter 1, I provide a structural and biochemical framework for understanding one of the key events of the initiation pathway: docking of the large (50S) ribosomal subunit with the small subunit 30S initiation complex (30S IC). The 50S subunit joining reaction is catalyzed by GTP-bound IF2 and results in formation of a 70S initiation complex (70S IC) that contains an initiator transfer RNA (tRNA) and is primed for formation of the first peptide bond. During 50S subunit joining, IF2-GTP establishes interactions with RNA and protein components of the 50S subunit’s GTPase-associated center (GAC), which play an important role in subunit recruitment as well as the subsequent activation of GTP hydrolysis by IF2. In Chapter 2, I describe the development of a single-molecule fluorescence resonance energy transfer (smFRET) signal to monitor the interactions between IF2 and the ribosome’s GAC during real-time 50S subunit joining reactions. Specifically, the role of the L11 region, comprising ribosomal protein L11 and its associated ribosomal RNA (rRNA) helices, was investigated. The L11 region is a prominent structural component of the GAC that is believed to undergo large-scale conformational changes during protein synthesis; however, the nature and timescale of these conformational dynamics, and their role in regulating the biochemical activities of IF2 during initiation, are not known. I demonstrate that my smFRET-based 50S subunit joining assay is sensitive to conformational rearrangements between IF2 and L11 within the 70S IC and can thus be used as a tool for characterizing GAC dynamics and elucidating their function during initiation. Furthermore, my smFRET approach is shown to provide information on the rate of 50S subunit joining as well as the rate of IF2 dissociation from the 70S IC. Notably, IF2-dependent GTP hydrolysis was found to influence the extent of 70S IC conformational dynamics as well as the dissociation rate of IF2. The role of IF3 in regulating 50S-subunit joining dynamics is discussed in Chapter 3. IF3 plays an important role in ensuring the fidelity of translation initiation by preventing the formation of initiation complexes containing a non-initiator tRNA and/or a non-canonical mRNA start codon. Inclusion of IF3 within the 30S IC in the smFRET experiments was found to render the IF2-catalyzed 50S subunit joining reaction highly reversible. Direct observation of repetitive docking and undocking of the 50S subunit with the 30S IC indicates that IF3 may modulate translation initiation efficiency by influencing the stability of the 70S IC. The individual 50S subunit docking events were found to result in the formation of very different classes of 70S IC, characterized by different stabilities and unique patterns of IF2-L11 interactions. I propose that these dynamics reflect an underlying conformational equilibrium of the IF3-bound 30S IC that is read out during 50S subunit joining, and that this equilibrium could be modulated in order to regulate the efficiency of translation initiation. Following initiation-factor mediated assembly of the 70S IC, the first aminoacyl- tRNA is delivered to the ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP. Accommodation of aminoacyl-tRNA into the ribosome’s peptidyl transferase center leads to formation of the first peptide bond, which signals the end of initiation and entry into the elongation phase of protein synthesis. The ternary complex binding site on the ribosome overlaps with that of IF2 at the GAC; a question of key mechanistic importance in understanding how the ribosome coordinates the transition from initiation to elongation thus concerns the relative timing of ternary complex binding with respect to IF2 dissociation from the 70S IC. In Chapter 4, I present preliminary results from two- and three-color fluorescence co-localization experiments aimed at characterizing the timing of these events at the single-molecule level. The data strongly suggest the occurrence of simultaneous occupancy of the ribosome by IF2 and ternary complex, implying that the ribosome is structurally capable of recruiting ternary complex prior to IF2 release from the 70S IC. The observation that the ribosome can accommodate more than one translation factor at a time may have important implications for understanding how it efficiently coordinates factor binding and release throughout protein synthesis, and opens the door to mechanistic studies of the ribosomal L7/L12 stalk, presumed to play a prominent role in these processes. Table of Contents List of Figures ................................................................................................................... vi List of Tables .................................................................................................................... ix List of abbreviations and acronyms .................................................................................x Acknowledgments .......................................................................................................... xiii Chapter 1 – Introduction ..................................................................................................1 1.1 Protein synthesis and the translational machinery ...................................................1 1.2 The prokaryotic translation initiation pathway ........................................................8 1.3 Structure and function of the initiation factors ......................................................10 1.3.1 Initiation Factor 1 (IF1) ................................................................................12 1.3.2 Initiation Factor 2 (IF2) ................................................................................12 1.3.3 Initiation Factor 3 (IF3) ................................................................................17 1.4 IF2-catalyzed 50S subunit joining .........................................................................21 1.5 Ribosome-dependent GTP hydrolysis by IF2 ........................................................25 1.5.1 Mechanism of GTP hydrolysis .....................................................................25 1.5.2 Role of GTP hydrolysis during initiation......................................................28 1.6 Interactions between IF2 and the GTPase-associated center .................................30 1.7 L11 structure and function .....................................................................................32 1.8 smFRET and TIRF microscopy: tools for studying dynamics ..............................39 1.9 Summary and motivation for my Ph.D. work ........................................................45 1.10 References ............................................................................................................48 Chapter 2 – Interaction of IF2 with the GTPase-associated center during 70S IC formation ..........................................................................................................................60 2.1 Introduction ............................................................................................................60 2.2 Design of smFRET probes .....................................................................................61 2.3 Generation of fluorescently labeled constructs ......................................................63 2.3.1 (Cy3/Cy5)-L11 50S subunits ........................................................................63 2.3.2 (Cy3/Cy5)-IF2...............................................................................................64 i 2.4 Biochemical testing of fluorescently labeled components.....................................66 2.4.1 GTP hydrolysis assay ....................................................................................66 fMet 2.4.2 Selection of fMet-tRNA on the 30S IC ...................................................69 2.4.3 Dipeptide formation assays ...........................................................................72 2.5 Characterization of L11-IF2 smFRET signals within 70SICGDPNP ........................75 2.6 Real-time observation of IF2-catalyzed 50S subunit joining ................................78 2.6.1 Development of experimental platform ........................................................78