RNA Catalysis and the Replication of Chromosome Telomeres

RNA was long thought to function solely as a genetic messenger, as a component of the ribosome, and as a carrier of amino acids. Now, largely because of research done at the University of Colorado, it is just as common to think of RNA participating directly in cellular catalysis. RNA can engage in intramolecular catalysis including self-splicing and in some cases can act as an enzyme. A major goal of the work carried out by Professor Cech and his research group is to understand mechanisms of RNA catalysis at both the chemical and biological levels. The work integrates organic and physical chemistry, enzymology, molecular biology, and structural biology.

Self-splicing group I introns, including one from the ciliated protozoan Tetrahymena, continue to be major experimental systems in the Cech laboratory. Although site-specific mutagenesis is still routinely used, semi-synthetic ribozymes now make it possible to change single atoms within very large RNA molecules. This enables study of the role of the nucleic acid backbone, not just the bases. A kinetic framework has been established and allows separation of the elemental steps of site-specific cleavage of RNA by the ribozyme. Thus, the effects of atomic substitutions on ribozyme folding, substrate binding, and the chemical cleavage step can be differentiated.

X-ray crystallography has proven invaluable for understanding structure-function relationships in proteins, but has been difficult to apply to large RNAs. Until recently, the largest RNAs for which crystal structures had been determined were transfer RNAs. In 1996 the Cech and Kundrot research groups, in collaboration with the group of Dr. Jennifer Doudna (a former postdoctoral fellow now on the faculty of Yale Univ.), succeeded in crystallizing and solving the structure of a 160-nucleotide domain of the Tetrahymena ribozyme. It was the first RNA structure large enough to show the level of RNA folding relevant to large catalytic RNAs: the side-by-side packing of helices, held together by tertiary base interactions, ribose-ribose hydrogen bonds and phosphate-metal ion interactions. In 1998 Barbara Golden, Anne Gooding and Elaine Podell in the Cech lab solved the first structure of an active group I ribozyme (247 nucleotides). It showed an active site largely preorganized for catalysis, much like a protein enzyme. These structures provide an excellent basis for planning future biochemical and X-ray crystallographic experiments.

The structure and replication of telomeres, the natural ends of linear chromosomes, provide a very different area of interest for a portion of the Cech group. Biological systems include Oxytricha, Euplotes, and Tetrahymena, ciliates with unusually large numbers of linear DNA molecules in their macronuclei and therefore high concentrations of telomere components. Publishing date: August 4, 2000

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