Scientific Report 2006

Molecular Biology

Nucleic Acid Dynamics

D.P. Millar, J. Gill, G. Pljevaljùci«c, S. Pond, G. Stengel, N. Tassew, 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 ribozymes, ribonucleoprotein complexes, and DNA polymerases. Our studies reveal the dynamic structural rearrangements that occur during the assembly and function of these macromolecular machines.

Ribozymes

RNA conformation plays a central role in the mechanism of ribozyme catalysis. The hairpin ribozyme is a small nucleolytic ribozyme that serves as a model system for studies of RNA folding and catalysis. The hairpin ribozyme consists of 2 internal loops, 1 of which contains the scissile phosphodiester bond, displayed on 2 arms of a 4-way multihelix junction.

To attain catalytic activity, the ribozyme must fold into a compact conformation in which the 2 loops become connected by a network of tertiary hydrogen bonds. We monitor the formation of this docked structure by using fluorescence resonance energy transfer (FRET) and ribozyme constructs labeled with donor and acceptor dyes within the loop-bearing arms. By measuring FRET at the level of single ribozyme molecules, we reveal subpopulations of compact and extended conformers that are not detected in ensemble experiments. Using this approach, we found that the ribozyme populates an intermediate state in which the 2 loops are in proximity but tertiary interactions have yet to form. This quasi-docked state forms rapidly (submillisecond timescale), but the subsequent formation of the tertiary contacts between the 2 loops occurs much more slowly. The hairpin ribozyme is an ideal system for exploring this fundamental mechanism of the formation of RNA tertiary structure.

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 the development of therapeutic drugs.

To dissect the mechanism of assembly of ribonucleoprotein complexes, we use single-molecule fluorescence imaging methods to monitor the progressive formation of oligomeric complexes of Rev on individual RRE molecules immobilized on a solid surface. We also use single-pair 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 binding of Rev to the RRE or prevent the subsequent Rev-Rev oligomerization.

DNA Polymerases

DNA polymerases are remarkable for their ability to synthesize DNA at rates approaching 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.

Our results reveal that binding of a correct nucleotide substrate induces a slow conformational change within the polymerase, causing the “fingers” subdomain to close over the DNA primer terminus and incoming nucleotide. Our studies are providing new insights into the dynamic structural changes responsible for nucleotide recognition and selection by DNA polymerases. Single-pair FRET methods are also being used to monitor the movement of the DNA primer/template between the separate polymerizing and editing sites of the enzyme. This active-site switching of DNA plays a key role in the proofreading process used to remove misincorporated nucleotides from the newly synthesized DNA. The advantage of single-molecule observations is that they eliminate the need to synchronize a population of molecules, allowing these dynamic processes to be directly observed.

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, in press.

Tian, F., Debler, E.W., Millar, D. P., Deniz, A.A., Wilson, I.A., Schultz, P.G. Multicolor fluorescent antibodies. Angew. Chemie, in press.