Single-molecule studies of conformational and compositional processes on the ribosome that regulate protein synthesis



Gene expression in the cell can be described as the set of regulated biochemical process that transmit information stored in DNA into specific RNA and protein molecules with structural and/or enzymatic function. The molecular processes underpinning gene expression are mediated by macromolecular assemblies which serve as manufacturing centers within the cell.

Complex, nanometer-scale enzyme assemblies called RNA polymerase and the ribosome are the principle players in the process of gene expression. In bacteria, the core RNA polymerase particle is a four subunit protein complex; the ribosome is two subunit RNA protein complex containing three RNA species and ~56 proteins. RNA polymerase first converts DNA into RNA in a process called transcription; the ribosome then converts RNA into protein in a process called translation. Transcription and translation are ubiquitous biological processes; RNA polymerase and the ribosome are highly conserved in primary sequence, architecture and function and operate with regulated rates and fidelities in the cell. Regulation is achieved through transient interactions with soluble factors whose interaction with RNA polymerase and the ribosome modulates the assembly conformation to affect template binding, substrate incorporation and directional motion.

The focus of my group is to develop novel single-molecule fluorescence methods that can be used to study conformational and compositional processes in translation that define both basal and regulated functions of the ribosome.

We have successfully reconstituted the process of translation in microfabricated fluid channels on a passivated quartz surface using components derived from the prokaryotic organism Escherechia coli, (E.coli). Purified, fluorescently-tagged ribosome particles are tethered near the quartz-liquid interface to allow continuous monitoring of single, spatially localized particles for up to several minutes (sufficient time to synthesize proteins). Factors and buffers are delivered to the immobilized ribosome using stopped-flow methods. Compositional and conformation processes within individual particles are monitored as the time-dependent evolution of fluorescence.

Using total internal reflection fluorescence microscopy (TIRFM) we aim to observe the principal enzymatic activities of the ribosome: transfer RNA selection, peptide-bond formation and directional translocation along messenger RNA template. To date we have focused on the use of fluorescence resonance energy transfer (FRET) to monitor the movement of tRNA through the ribosome during translation. In our most recent work, we have shown that we can directly observe the process of tRNA selection on the ribosome. aa-tRNA molecules incorporate into the particle through a series of step-wise movements. Several antibiotics and non-hydrolyzable GTP analogues disrupt the normal process of movement between steps. We are currently using this system to define the kinetic parameters of aa-tRNA selection using statistical methods and the parameters that dictate fidelity in this process. In doing so, we hope to understand the molecular mechanism by which the ribosome is able to accurately choose the correct tRNA during protein synthesis and to understand the manner by which antibiotics effect this process.

e-mail: scb2005@med.cornell.edu

For further information please visit: http://physiology.med.cornell.edu/faculty/blanchard/index.html