Gene expression and its control are inextricably tied to every biochemical event in all living cells. As such, deregulation of gene expression is associated with numerous human diseases, most notably cancer and viral infections. Gene expression and its regulation are mediated by complex biomolecular assemblies known as RNA polymerase, the spliceosome and the ribosome. These act as molecular motors in the biochemical processes of transcription, splicing and protein synthesis respectively. Our research focuses on the thermodynamics, kinetics and structures of these biomolecular assemblies and molecular motors, with specific emphasis on the ribosome and protein synthesis.
As a molecular motor, the ribosome and its substrates undergo numerous highly-coordinated, transient molecular motions during protein synthesis; the process is necessarily dynamic. Nevertheless, dynamic information is lacking in current mechanistic models of protein synthesis. For example, the sequence, cooperativity and lifetimes of substrate binding events are poorly defined. In addition, the transient dynamic nature of crucial structural intermediates has impeded study of their role in protein synthesis. Coordinated conformational rearrangements of the ribosome and its substrates, possibly important for allosteric signaling, have also not been kinetically characterized. Research addressing these dynamic aspects is critical for establishing a complete mechanistic model and providing a full understanding of protein synthesis and its regulation. To explore the dynamics of protein synthesis, we take a variety of biochemical, kinetic and structural approaches, making particularly extensive use of single-molecule fluorescence spectroscopy.
The kinetics of substrate binding in a complex biochemical reaction can be easily followed using fluorescently labeled substrates and single-molecule fluorescence. Also, single-molecule fluorescence resonance energy transfer (smFRET) is uniquely suited to bridge the gaps between static structure, conformational dynamics and biochemical function. The efficiency of smFRET depends on the distance between a donor and acceptor dye-pair and can therefore be used to monitor conformational changes by measuring changes in distance between donor and acceptor dyes as a function of time. By eliminating ensemble-averaging, single-molecule experiments allow study of the distributions and time trajectories of physical properties, such as composition and conformational state, which would ordinarily remain masked in bulk. In addition, biochemical reactions that are difficult or impossible to synchronize when studied in bulk can be effectively studied using single-molecule experiments. Finally, single-molecule fluorescence allows investigation of kinetics on a milliseconds timescale, matching the expected timescale of conformational rearrangements of the ribosome and its substrates.
A complete mechanistic understanding of the ribosomal molecular machine and its role in carrying out and regulating protein synthesis will serve as a paradigm for understanding related molecular machines and biomolecular assemblies involved in other aspects of gene expression and its regulation.