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Thomas R Cech

Publications and source records attributed to Thomas R Cech.

40 records · Page 3Linked to original sources
PubMed2002

Evolution of Tetrahymena ribozyme mutants with increased structural stability.

Determining how large RNA molecules stabilize their tertiary structures is critical for understanding how they perform their biological functions. Here we use in vitro selection to identify active variants of the Tetrahymena ribozyme with increased stability. The mutant pool converged to a single family that shared nine mutations; an RNA representing the consensus sequence was structurally more stable by 10.5 degrees C and catalytically active at elevated temperatures. Remarkably, of the nine altered sites, most are already known to be involved in tertiary interactions, and the stabilizing mutations primarily improve the packing interactions in the molecular interior. The wild type ribozyme and the selected mutants provide pairs of mesophilic and thermophilic homologs for studying the origin of their thermal stability.

Animals↗
PubMed2002

Human Pot1 (protection of telomeres) protein: cytolocalization, gene structure, and alternative splicing.

Fission yeast Pot1 (protection of telomeres) is a single-stranded telomeric DNA binding protein with a critical role in ensuring chromosome stability. A putative human homolog (hPot1) was previously identified, based on moderate sequence similarity with fission yeast Pot1 and telomere end-binding proteins from ciliated protozoa. Using indirect immunofluorescence, we show here that epitope-tagged hPot1 localizes to telomeres in interphase nuclei of human cells, consistent with a direct role in telomere end protection. The hPOT1 gene contains 22 exons, most of which are present in all cDNAs examined. However, four exons are subject to exon skipping in some transcripts, giving rise to five splice variants. Four of these are ubiquitously expressed, whereas the fifth appears to be specific to leukocytes. The resultant proteins vary significantly in their ability to form complexes with single-stranded telomeric DNA as judged by electrophoretic mobility shift assays. In addition to these splice variants, the Pot1 family is expanded by the identification of six more genes from diverse species. Pot1-like proteins have now been found in plants, animals, yeasts, and microsporidia.

Alternative Splicing↗
PubMed2002

Essential regions of Saccharomyces cerevisiae telomerase RNA: separate elements for Est1p and Est2p interaction.

The Saccharomyces cerevisiae telomerase RNA subunit is encoded by the TLC1 gene. A selection for viable alleles of TLC1 RNA from a large library of random deletion alleles revealed that less than half (approximately 0.5 kb of the approximately 1.3-kb RNA) is required for telomerase function in vivo. The main essential region (430 nucleotides), which contains the template for telomeric DNA synthesis, was required for coimmunoprecipitation with Est1p and Est2p. Furthermore, the subregion required for interaction with Est1p, the telomerase recruitment subunit, differed from those required for interaction with Est2p, the reverse transcriptase subunit. Two regions of the RNA distant from the template in the nucleotide sequence were required for Est2p binding, but the template itself was not. Having the RNA secured to the protein away from the template is proposed to facilitate the translocation of the RNA template through the active site. More generally, our results support a role for the telomerase RNA serving as a scaffold for binding key protein subunits.

Alleles↗