Single-molecule FRET-Rosetta reveals RNA structural rearrangements during human telomerase catalysis.

Maintenance of telomeres by telomerase permits continuous proliferation of rapidly dividing cells, including the majority of human cancers. Despite its direct biomedical significance, the architecture of the human telomerase complex remains unknown. Generating homogeneous telomerase samples has presented a significant barrier to developing improved structural models. Here we pair single-molecule Förster resonance energy transfer (smFRET) measurements with Rosetta modeling to map the conformations of the essential telomerase RNA core domain within the active ribonucleoprotein. FRET-guided modeling places the essential pseudoknot fold distal to the active site on a protein surface comprising the C-terminal element, a domain that shares structural homology with canonical polymerase thumb domains. An independently solved medium-resolution structure of Tetrahymena telomerase provides a blind test of our modeling methodology and sheds light on the structural homology of this domain across diverse organisms. Our smFRET-Rosetta models reveal nanometer-scale rearrangements within the RNA core domain during catalysis. Taken together, our FRET data and pseudoatomic molecular models permit us to propose a possible mechanism for how RNA core domain rearrangement is coupled to template hybrid elongation.

An assessment of proteolytic enzymes in Tetrahymena thermophila.

Cellular extracts of Tetrahymena thermophila were found to contain substantial levels of proteolytic activity. Protein digestion occurred over broad ranges of pH, ionic strength, and temperature and was stimulated by treatment with thiol reductants, EDTA and sodium dodecyl sulfate. Incubation at temperatures > or = 60 degrees C or with high concentrations of chaotropic reagents such as 10 M urea or 6 M guanidine-HCl caused an apparent irreversible loss of activity. Activity was also strongly diminished by increasing concentrations of divalent cations. Several peptide aldehydes, p-hydroxymercuribenzoate, and alkylating reagents such as iodoacetate, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, N-methylmaleimide, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane were potent inhibitors of proteolytic activity. Aprotinin diminished activity by approximately 40% while benzamidine, 3,4-dichlorosocoumarin, and trypsin inhibitors from soy bean, lima bean, and chicken egg caused relatively modest inhibition of proteolytic activity. Phenylmethanesulfonyl fluoride had no apparent effect. Electrophoretic separation of proteins on SDS-polyacrylamide gels copolymerized with gelatin substrate revealed that at least eight active proteolytic enzymes were present in cell extracts ranging in apparent molecular weight from 45,000 to 110,000. Five of these apparent proteases were detected in 70% ammonium sulfate precipitates. Gelatinase activity was not detectable when extracts were pretreated with iodoacetate or E-64, indicating that all of the enzymes observed in activity gels were sensitive to thiol alkylation. Cellular extracts of T. thermophila appeared to contain multiple forms of proteolytic enzymes which were stimulated by thiol reductants and inhibited by thiol modifying reagents. Accordingly, the proteolytic enzymes present in cell extracts appear to be predominantly cysteine proteinases.