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Enzymatic RNA Molecules and the Replication of Chromosome Ends
Summary: Tom Cech and his group are working to understand the structure and function of catalytic RNA molecules and the activity and regulation of telomerase.
A cell must orchestrate thousands of chemical reactions in order to live, to grow, and to respond to its environment. These chemical reactions rarely happen spontaneously but are usually catalyzed by macromolecules called enzymes. It was long thought that all enzymes were proteins. More recently we and others have found that RNA can in some cases act as an enzyme.
The finding of RNA catalysis has several implications. First, it means that RNA is not solely a passive carrier of genetic information but can participate actively in directing cellular biochemistry. In particular, many RNA-processing reactions, as well as protein synthesis on ribosomes, are at least in part catalyzed by RNA. Second, the study of how RNA enzymes work may reveal hitherto unknown mechanisms of biologic catalysis. Third, RNA enzymes (ribozymes) have the potential to provide new therapeutic agents. For example, ribozymes efficiently cleave and thereby destroy viral RNAs under controlled laboratory conditions, making it plausible that ribozymes might be able to inactivate viruses in animals, including people.
Many of our studies of RNA catalysis concern the Tetrahymena ribozyme, named for the single-celled animal from which it was originally isolated. This RNA enzyme is capable of cleaving other RNA molecules (substrates) in a sequence-specific manner. Our main objective is to understand the mechanism by which this RNA molecule acts as a catalyst, in part by obtaining a high-resolution structure of its active site.
X-Ray Crystallography of RNA
In 1998, a major step was taken toward the goal of obtaining a detailed picture of a ribozyme catalytic center. The 5.0-Å crystal structure of a 247-nucleotide, fully active form of the Tetrahymena ribozyme was solved by Barbara Golden, Elaine Podell, and Anne Gooding in our laboratory. It revealed an active-site cleft large enough to fit the RNA helix (called P1, for paired region 1) that is cleaved by the ribozyme. We concluded that this RNA enzyme is largely preorganized for substrate binding and catalysis, much like a typical protein enzyme. In both cases, the macromolecule usually undergoes some conformational adjustment to bind the substrates, but its global architecture is maintained.
How can we obtain a higher-resolution, atomic-level view of an active ribozyme? One promising approach has been developed by Feng Guo, an HHMI associate. He has used "evolution in the test tube" to identify subtle mutations in the Tetrahymena ribozyme that cause it to fold more stably. We had hoped that more-stable folding would correlate with better crystallization properties, and this now appears to be the case.
Replication of Chromosome Ends by Telomerase
Telomerase is the key enzyme for replication of the ends of linear chromosomes, such as those found in human cells. Its absence has been implicated in cellular aging, and its reactivation promotes tumorigenesis. Telomerase is an unusual enzyme in that it contains both essential RNA and protein components. In 1996, work by Joachim Lingner (then an HHMI associate in the laboratory) led to the discovery of the catalytic protein subunit of telomerase in Euplotes aediculatus. Computer searching and biochemical approaches then allowed the genes for the corresponding telomerase subunits to be identified in other protozoa, including Tetrahymena, budding and fission yeast, and humans. These proteins comprise a new family of telomerase reverse transcriptases (TERTs), distant relatives of the enzyme responsible for copying the RNA of the human immunodeficiency virus, HIV.
Our group is now focusing on the assembly, structure, and function of telomerase, with special emphasis on the roles of the RNA subunit. Most of our work is carried out in budding yeast or fission yeast, which are amenable to biochemistry but also allow interrogation of function in vivo. Recent research is aimed in part at understanding structure-function relationships in the Saccharomyces cerevisiae telomerase RNP. We have developed a working model of the RNA secondary structure on the basis of free-energy computation, comparative analysis, and site-specific mutagenesis. The RNA appears to have three quasi-helical "arms" emanating from a central core. The core contains the template and the TERT-binding site, while the ends of the three arms bind the regulatory subunit Est1p, the Ku heterodimer involved in DNA repair, and the Sm proteins involved in RNP maturation, respectively. Experiments indicate that the RNA provides flexible tethers for these proteins, rather than a precise scaffold. This is very different from other well-studied RNPs such as the ribosome. (A grant from the National Institutes of Health provides support for our telomerase projects.)
The Protein That Caps the Chromosome End
Telomerase activity at the telomere is regulated in large part by proteins that bind to its telomeric DNA substrate. In 1990, we reported the cloning and sequencing of the genes for the heterodimeric telomere end-binding protein (TEBP) from the ciliate Oxytricha and the reconstitution of the TEBP–single-stranded DNA ternary complex using recombinant proteins. We later discontinued research on these proteins, however, because it appeared that they might be unique to the ciliated protozoa. It thus came as a pleasant surprise in 2001 when Peter Baumann in the lab found homologs of the α subunit of TEBP in Schizosaccharomyces pombe and human. We named these proteins Pot1, for "protection of telomeres." S. pombe Pot1p is essential for telomere maintenance and therefore represents an indispensable chromosome cap. Human Pot1 protein colocalizes with two other known telomeric proteins in the nuclei of tissue culture cells, providing cytological confirmation of its identification. Both proteins, as well as their DNA-binding domains, have been expressed and purified. Each binds to its cognate telomeric DNA sequence with high sequence specificity and with a preference for the 3' end relative to internal sites on the single-stranded DNA.
Ming Lei and Elaine Podell in our lab recently solved the crystal structure of the DNA-binding domain of the pombe Pot1 protein bound to its cognate single-stranded DNA. The structure reveals a novel feature, "DNA self-recognition," that explains the molecular basis of telomeric DNA sequence recognition. The discovery and characterization of these Pot1 proteins pave the way for better understanding chromosome capping and telomerase regulation in diverse eukaryotes, including humans.
Last updated: May 15, 2007
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