Functional Interactions of Kluyveromyces lactis Telomerase Reverse Transcriptase with the Three-Way Junction and the Template Domains of Telomerase RNA.

The ribonucleoprotein telomerase contains two essential components: telomerase RNA (TER) and telomerase reverse transcriptase (TERT, Est2 in yeast). A small portion of TER, termed the template, is copied by TERT onto the chromosome ends, thus compensating for sequence loss due to incomplete DNA replication and nuclease action. Although telomerase RNA is highly divergent in sequence and length across fungi and mammals, structural motifs essential for telomerase function are conserved. Here, we show that Est2 from the budding yeast Kluyveromyces lactis (klEst2) binds specifically to an essential three-way junction (TWJ) structure in K. lactis TER, which shares a conserved structure and sequence features with the essential CR4-CR5 domain of vertebrate telomerase RNA. klEst2 also binds specifically to the template domain, independently and mutually exclusive of its interaction with TWJ. Furthermore, we present the high-resolution structure of the klEst2 telomerase RNA-binding domain (klTRBD). Mutations introduced in vivo in klTRBD based on the solved structure or in TWJ based on its predicted RNA structure caused severe telomere shortening. These results demonstrate the conservation and importance of these domains and the multiple protein-RNA interactions between Est2 and TER for telomerase function.

High-temperature mass spectrometric study of thermodynamic properties in the TiO2 -Al2 O3 -SiO2 system and modeling.

RATIONALE: The TiO2 -Al2 O3 -SiO2 system is the base for various glass-ceramic materials, which have great practical value for a large number of modern technologies. Many TiO2 -Al2 O3 -SiO2 materials are synthesized or applied at high temperatures, which justifies the relevance of the present study. METHODS: The samples in the TiO2 -Al2 O3 -SiO2 system were synthesized using the method of induction melting in a cold crucible. The thermodynamic properties of the TiO2 -Al2 O3 -SiO2 system were studied using the Knudsen effusion mass spectrometric method. The derived thermodynamic functions were optimized within the generalized lattice theory of associated solutions (GLTAS) approach and compared with the results of calculation using the semiempirical Kohler, Muggianu, Toop, Redlich-Kister, and Wilson methods based on the corresponding data in the binary systems. RESULTS: The SiO2 selective vaporization from the samples under study was shown at temperatures above 1940 K. The thermodynamic properties in the TiO2 -Al2 O3 -SiO2 system, including the TiO2 -SiO2 system, were obtained in the temperature range 1965-2012 K and were optimized using GLTAS to obtain the consistent concentration dependences of the component activities and excess Gibbs energies. CONCLUSIONS: Positive deviations from the ideal behavior were observed in the TiO2 -Al2 O3 -SiO2 system at high temperatures. Comparison of these values with the results of the modeling based on the GLTAS approach allowed the recommendations regarding the optimal semiempirical methods for the excess Gibbs energy calculation in different concentration ranges to be made.

A motif in the vertebrate telomerase N-terminal linker of TERT contributes to RNA binding and telomerase activity and processivity.

Telomerase is a ribonucleoprotein reverse transcriptase that replicates the ends of chromosomes, thus maintaining genome stability. Telomerase ribonucleoprotein assembly is primarily mediated by the RNA binding domain (TRBD) of the enzyme. Here we present the high-resolution TRBD structure of the vertebrate, Takifugu rubripes (trTRBD). The structure shows that with the exception of the N-terminal linker, the trTRBD is conserved with the Tribolium castaneum and Tetrahymena thermophila TRBDs, suggesting evolutionary conservation across species. The structure provides a view of the structural organization of the vertebrate-specific VSR motif that binds the activation domain (CR4/5) of the RNA component of telomerase. It also reveals a motif (TFLY) that forms part of the T-CP pocket implicated in template boundary element (TBE) binding. Mutant proteins of conserved residues that consist of part of the T and TFLY motifs disrupt trTRBD-TBE binding and telomerase activity and processivity, supporting an essential role of these motifs in telomerase RNP assembly and function.