Scientific Report 2008

Molecular Biology

Nucleic Acid Dynamics

D.P. Millar, J. Gill, G. Pljevaljci´c, R. Robertson, J. Wang, E.J.C. Van der Schans

The focus of our research is the assembly and conformational dynamics of nucleic acid—based macromolecular machines and assemblies. We use single-molecule fluorescence methods to investigate a range of systems, including ribonucleoprotein complexes and DNA polymerases. Our studies reveal the dynamic structural rearrangements that occur during the assembly and function of these complex macromolecular assemblies.

Ribonucleoprotein Assembly

The Rev protein from HIV type 1 is a key regulatory protein that controls the transition from early to late patterns of viral gene expression. Rev binds to a highly structured region within the viral mRNA, known as the Rev response element (RRE), where it forms an oligomeric ribonucleoprotein complex. The formation of this complex inhibits splicing and facilitates export of the viral RNA from the nucleus to the cytoplasm. Because of its critical role in the viral life cycle, the Rev-RRE complex provides a novel target for development of therapeutic drugs.

To dissect the mechanism of assembly of ribonucleoproteins, we use single-molecule fluorescence imaging methods to monitor the formation of oligomeric complexes of Rev on individual RRE molecules immobilized on a solid surface. We found that a single Rev monomer binds initially to a high-affinity site in stem loop IIB of the RRE and that assembly subsequently proceeds by the stepwise addition of additional Rev monomers. The elementary rate constants for each step of assembly were also obtained from the single-molecule data. We also use single-pair Förster or fluorescence resonance energy transfer (FRET) to probe changes in the conformation of the RRE during the assembly process. We are using the results of these mechanistic studies to develop novel fluorescence-based methods for high-throughput screening of libraries of chemical compounds. The new screening tools are being used to identify small molecules that block initial binding of Rev to the RRE or prevent the subsequent Rev-Rev oligomerization.

Another example of ribonucleoprotein assembly under study is the signal recognition particle. This particle is a fascinating molecular machine responsible for the cotranslational targeting of secretory or membrane proteins to the endoplasmic reticulum. This large complex, composed of a 300-nucleotide RNA and 6 proteins, interacts with both the ribosome, during translational arrest, and a membrane-bound receptor. We are developing novel spectroscopic techniques (based on multicolor FRET) to dissect the assembly pathway of the particle, focusing on the temporal order of protein-binding events and the associated RNA-folding transitions.

In parallel, we are developing methods to label large RNA molecules with donor and acceptor probes for FRET measurements. These reagents and methods are being used to monitor assembly reactions of signal recognition particle proteins on individual immobilized RNA molecules by means of single-molecule FRET microscopy. The interplay between protein-binding and RNA-folding events revealed in our studies is providing general insights into the mechanism of assembly of ribonucleoproteins.

DNA Polymerases

DNA polymerases are remarkable for their ability to synthesize DNA at rates of several hundred base pairs per second while maintaining an extremely low frequency of errors. To elucidate the origin of polymerase fidelity, we are using single-molecule fluorescence methods to examine the dynamic interactions that occur between a DNA polymerase and its DNA and nucleotide substrates. The FRET method is being used to observe conformational transitions of the enzyme-DNA complex that occur during selection and incorporation of an incoming nucleotide substrate. Similar methods are being used to monitor the proofreading step after synthesis.

Our results reveal that binding of a correct nucleotide substrate induces a slow conformational change within the polymerase, causing the "fingers” domain to close over the DNA primer terminus and incoming nucleotide. Our studies are also providing new insights into the mechanisms used to transfer a DNA substrate from the synthesis site to the exonuclease active site during proofreading. The advantage of single-molecule observations is that they eliminate the need to synchronize a population of molecules, allowing these dynamic processes to be observed directly.

Publications

Bailey, M.F., Van der Schans, E.J.C., Millar, D.P. Dimerization of the Klenow fragment of Escherichia coli DNA polymerase I is linked to its mode of DNA binding. Biochemistry 46:8085, 2007.

Debler, E.W., Kaufmann, G.F., Meijler, M.M., Heine, A., Mee, J.M., Pljevaljci´c, G., Di Bilio, A.J., Schultz, P.G., Millar, D.P., Janda, K.D., Wilson, I.A., Gray, H.B., Lerner, R.A. Deeply inverted electron-hole recombination in an antibody-stilbene complex. Science 319:1232, 2008.

Pljevaljci´c, G., Millar, D. Single-molecule fluorescence methods for the analysis of RNA folding and ribonucleoprotein assembly. Methods Enzymol., in press.

Stengel, G., Gill, J.P., Sandin, P., Wilhelmsson, M., Albinsson, B., Nordén, B., Millar, D. Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue. Biochemistry 46:12289, 2007.