Tinoco Research Group

Department of Chemistry University of California, Berkeley

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Research: Physical Chemistry of Nucleic Acids

The conformations of nucleic acids depend on their base sequences, and on their environments. The sequences and conformations determine their biological functions. We want to understand all of this. Current projects include:

Single-molecule studies of RNA

We are also studying how RNA molecules respond to local application of mechanical force. Currently, we are investigating the T. Thermophila ribozyme, various small hairpins, and ribosomal RNA. A single RNA is held between beads and force is applied to unfold it in any chosen condition. The work done in unfolding the RNA provides the free energy of the reaction in the chosen environment. The kinetics of the reaction can also be obtained. We have recently started studies of protein translation by individual ribosomes. Translation occurs by a series of steps with translocations of approximately 0.1 s followed by pauses of order 1 s. We are learning the effects of messenger RNA sequence and structure, and of antibiotics, on translation, in particular on the mechanism of frameshifting.

Determination of the conformations of important structural elements in RNA

RNA is synthesized as a linear polynucleotide that folds into its biolgically functional form. It can be studied as a combination of structural elements: double helices, hairpins, internal loops, bulges and triple-base-interactions. We are systematically determining the structures of these elements and measuring the thermodynamics of their formation from single-stranded RNA. The main methods we use are two- and three- dimensional NMR experiments involving 1H, 13C, 15N and 31P nuclei to determine structures, and single-molecule force vs. extension experiments to determine thermodynamics. Other methods include circular dichroism, fluorescence, reactivity to chemical probes and to single-strand and double-strand specific enzymes, and any other technique that works.

Determination of the conformations of RNA pseudoknots, kissing hairpins, and metal-ion binding sites

A pseudoknot is a form of tertiary structure that occurs often in RNA and is involved in regulation of transcription and translation. Kissing hairpins occur when the loops of two RNA hairpins form base pairs. This is the first step in natural antisense control of RNA biological function. Metal ions are vital to the function of RNA molecules. Cobalt (III) hexammine can substitute for the biologically-relevant magnesium hexahydrate in RNA binding sites. We are determining the structures and thermodynamics of these RNA motifs, and we are learning how they are affected by binding to proteins and drugs.

Characterization of catalytic RNA

RNA molecules have the ability to catalyze chemical reactions, including the hydrolysis and ligation of specific phosphodiester bonds. We are studying the conformations and kinetics of different catalytic RNA molecules (ribozymes), with the main emphasis on the group I ribozyme from Tetrahymena thermophila. The general questions are: how does RNA fold to pro duce a catalytic site, and what is the mechanism of the catalysis?