Scientific Report 2005

Molecular Biology

Single-Molecule Conformational Dynamics of Nucleic Acid Enzymes

D.P. Millar, M.F. Bailey, G. Pljevaljčić, 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. We use single-molecule fluorescence methods to investigate a range of systems, including ribozymes, DNA polymerases, and topoisomerases. Our studies reveal the large structural rearrangements that occur as an integral component of the catalytic mechanism of these enzymes.

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 detailed biophysical 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 specific conformation in which the 2 loops are docked with each other, forming 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. By measuring FRET at the level of single ribozyme molecules, we reveal subpopulations of compact and extended conformers that are hidden in conventional 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 time scale), but the subsequent formation of tertiary contacts between the loops occurs much more slowly. Surprisingly, the rate of formation of tertiary structure is essentially independent of temperature, indicating that the activation enthalpy is negligible. Hence, the slow tertiary folding is due to an unfavorable entropy change in reaching the transition state.

These observations reveal that the tertiary structure of the hairpin ribozyme is formed through a slow conformational search process. This fundamental mechanism of formation of RNA tertiary structure was obscured in most previous folding studies because of the strong propensity of RNA molecules to populate nonnative conformations that act as kinetic traps during the course of folding.

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, altering the contacts between the enzyme and the DNA primer/template. This conformational change appears to primarily involve the finger and thumb subdomains of the enzyme. 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.

Topoisomerases

Topoisomerases are enzymes that control the state of DNA supercoiling in the cell. Type I topoisomerases introduce a nick into a strand of DNA and become covalently joined to the cleaved strand. This process allows the other strand to freely swivel around the first, resulting in the relaxation of supercoils within the DNA. The enzyme-DNA connection is then reversed, and the broken strand is rejoined, completing the process of supercoil removal. We are using single-pair FRET methods to observe the DNA-unwinding activity of single type I topoisomerase enzymes in real time. The purpose of these studies is to directly observe DNA rotational motions during supercoil relaxation and to determine whether the same number of supercoils is removed during each enzyme-DNA encounter.

Publications

Millar, D., Traskelis, M.A., Benkovic, S.J. On the solution structure of the T4 sliding clamp (gp45). Biochemistry 43:12723, 2004.

Pljevaljčić, G., Klostermeier, D., Millar, D.P. The tertiary structure of the hairpin ribozyme is formed through a slow conformational search. Biochemistry 44:4870, 2005.