Analysis of telomerase catalytic subunit mutants in vivo and in vitro in Schizosaccharomycespombe.

The chromosome end-replicating enzyme telomerase is composed of a template-containing RNA subunit, a reverse transcriptase (TERT), and additional proteins. The importance of conserved amino acid residues in Trt1p, the TERT of Schizosaccharomyces pombe, was tested. Mutation to alanine of the proposed catalytic aspartates in reverse transcriptase motifs A and C and of conserved amino acids in motifs 1 and B' resulted in defective growth, progressive loss of telomeric DNA, and loss of detectable telomerase enzymatic activity in vitro. Mutation of the phenylalanine (F) in the conserved FYxTE of telomerase-specific motif T had no phenotype in vivo or in vitro whereas mutation of a conserved amino acid in RT motif 2 had an intermediate effect. In addition to identifying single amino acids of TERT required for telomere maintenance in the fission yeast, this work provides useful tools for S. pombe telomerase research: a functional epitope-tagged version of Trt1p that allows detection of the protein even in crude cellular extracts, and a convenient and robust in vitro enzymatic activity assay based on immunopurification of telomerase.

Two modes of survival of fission yeast without telomerase.

Deletion of the telomerase catalytic subunit gene trt1+ in Schizosaccharomyces pombe results in death for the majority of cells, but a subpopulation survives. Here it is shown that most survivors have circularized all of their chromosomes, whereas a smaller number maintain their telomeres presumably through recombination. When the telomeric DNA-binding gene taz1+ is also deleted, trt1- taz1- survivors use the recombinational mode more frequently. Moreover, the massive elongation of telomeres in taz1- cells is absent in the double mutant. Thus, Taz1p appears to regulate telomeric recombination as well as telomerase activity in fission yeast.

Telomerase catalytic subunit homologs from fission yeast and human.

Catalytic protein subunits of telomerase from the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs. Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified. Disruption of the S. pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines. Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives. Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases.

Telomerase is a true reverse transcriptase. A review.

Synthesis of telomeric repeats at chromosome ends requires telomerase, a ribonucleoprotein enzyme. The RNA subunit, which contains the template for DNA synthesis, has been identified in many organisms. Recently, the protein subunit that catalyzes telomeric DNA extension has also been identified in Euplotes aediculatus and Saccharomyces cerevisiae. It has sequence and functional characteristics of a reverse transcriptase related to retrotransposon and retroviral reverse transcriptases, so this new family of telomerase subunits has been named TRT (Telomerase Reverse Transcriptase). We find it remarkable that the same type of protein structure required for retroviral replication is now seen to be essential for normal chromosome telomere replication in diverse eukaryotes.

Relative orientation of RNA helices in a group 1 ribozyme determined by helix extension electron microscopy.

The relative orientation of helical elements in a folded RNA molecule provides key information about its three-dimensional architecture. We have developed a method that involves extending peripheral helices of an RNA, mounting for electron microscopy in the absence of protein and measuring interhelical angles. As a control, extended anticodon and acceptor stems of tRNA(Phe) were found to form a 92 +/- 20 degrees angle, consistent with the X-ray structure. Single, double and triple extensions (50-80 bp) of helical elements P2.1, P6b and P8 of the Tetrahymena group I ribozyme did not alter its catalytic activity. The measured angle between P6b and P8 is consistent with the Michel-Westhof structural model, while the P2.1-P6b and P2.1-P8 angles allow P2.1 to be positioned in the model. The angle distributions of the ribozyme are broader than those of the tRNA, which may reflect the dynamics of the RNA. Helix extension allows low-resolution electron microscopy to provide much higher resolution information about the disposition of helical elements in RNA. It should be applicable to diverse RNAs and ribonucleoprotein complexes.