Interaction of human telomerase with its primer substrate

Telomerase is a ribonucleoprotein responsible for maintaining the ends of linear chromosomes in nearly all eukaryotic cells. In humans, expression of the enzyme is limited primarily to the germ line and progenitor cell populations. In the absence of telomerase activity, telomeres shorten with each cell division until a critical length is reached, which can result in the cessation of cell division. The enzyme is required for cell immortality, and its activity has been detected in the vast majority of human tumors. Because of this, telomerase is an attractive target for inhibition in anticancer therapy. To learn more about the biochemistry of the human enzyme and its substrate recognition, we have examined the binding properties of single-stranded oligonucleotide primers that serve as telomerase substrates in vitro. We have used highly purified human enzyme and employed a two-primer method for determining the dissociation rates of these primers. Primers having sequence permutations of (TTAGGG)(3) were found to have considerably different affinities. They had t(1/2) values that ranged from 14 min to greater than 1200 min at room temperature. A primer ending in the GGG register formed the most stable complex with the enzyme. This particular register imparted stability to a nontelomeric primer resulting in a nearly 100-fold decrease in the k(off). We have found that interactions of telomerase with primer substrates are stabilized mainly by contacts with the protein subunit of the enzyme (hTERT). Base-pairing between the primer and the template region of telomerase contributes minimally to its stabilization.     

A high-throughput assay for a human telomerase protein-human telomerase RNA interaction

The rapid rate at which cancer cells divide necessitates a mechanism for telomere maintenance, and in approximately 90% of all cancer types the enzyme telomerase is used to maintain the length of telomeric DNA. Telomerase is a multi-subunit enzyme that minimally contains a catalytic protein subunit, hTERT, and an RNA subunit, hTR. Proper assembly of telomerase is critical for its enzymatic activity and therefore is a requirement for the proliferation of most cancer cells. We have developed the first high-throughput screen capable of identifying small molecules that specifically perturb human telomerase assemblage. The screen uses a scintillation proximity assay to identify compounds that prevent a specific and required interaction between hTR and hTERT. Rather than attempting to disrupt all of the individual hTR-hTERT interactions, we focused the screen on the interaction of the CR4-CR5 domain of hTR with hTERT. The screen employs a biotin-labeled derivative of the CR4-CR5 domain of hTR that independently binds [(35)S]hTERT in a functionally relevant manner. The complex between hTERT and biotin-labeled RNA can be captured on streptavidin-coated scintillation proximity beads. Use of 96-well filter plates and a vacuum manifold enables rapid purification of the beads. After optimization, statistical evaluation of the screen generated a Z' factor of 0.6, demonstrating the high precision of the assay.     

A single-molecule assay for telomerase structure-function analysis

The activity of the telomerase ribonucleoprotein enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures.     

A conserved secondary structure for telomerase RNA.

The RNA moiety of the ribonucleoprotein enzyme telomerase contains the template for telomeric DNA synthesis. We present a secondary structure model for telomerase RNA, derived by a phylogenetic comparative analysis of telomerase RNAs from seven tetrahymenine ciliates. The telomerase RNA genes from Tetrahymena malaccensis, T. pyriformis, T. hyperangularis, T. pigmentosa, T. hegewishii, and Glaucoma chattoni were cloned, sequenced, and compared with the previously cloned RNA gene from T. thermophila and with each other. To define secondary structures of these RNAs, homologous complementary sequences were identified by the occurrence of covariation among putative base pairs. Although their primary sequences have diverged rapidly overall, a strikingly conserved secondary structure was identified for all these telomerase RNAs. Short regions of nucleotide conservation include a block of 22 totally conserved nucleotides that contains the telomeric templating region.     

Lasker Laurels for telomerase

This year the Lasker Foundation pays tribute to telomerase, a medically important enzyme required for chromosome stability and long-term cell proliferation.     

A Cajal body-independent pathway for telomerase trafficking in mice

The intranuclear trafficking of human telomerase involves a dynamic interplay between multiple nuclear sites, most notably Cajal bodies and telomeres. Cajal bodies are proposed to serve as sites of telomerase maturation, storage, and assembly, as well as to function in the cell cycle-regulated delivery of telomerase to telomeres in human cells. Here, we find that telomerase RNA does not localize to Cajal bodies in mouse cells, and instead resides in separate nuclear foci throughout much of the cell cycle. However, as in humans, mouse telomerase RNA (mTR) localizes to subsets of telomeres specifically during S phase. The localization of mTR to telomeres in mouse cells does not require coilin-containing Cajal bodies, as mTR is found at telomeres at similar frequencies in cells from wild-type and coilin knockout mice. At the same time, we find that human TR localizes to Cajal bodies (as well as telomeres) in mouse cells, indicating that the distinct trafficking of mTR is attributable to an intrinsic property of the RNA (rather than a difference in the mouse cell environment such as the properties of mouse Cajal bodies). We also find that during S phase, mTR foci coalesce into short chains, with at least one of the conjoined mTR foci co-localizing with a telomere. These findings point to a novel, Cajal body-independent pathway for telomerase biogenesis and trafficking in mice.     

A new chromogenic substrate for subtilisin

A new chromogenic substrate, benzyloxycarbonyl-glycyl-glycyl-l-leucine p-nitroanilide, was synthesiszed allowing convenient spectrophotometric assay of subtilisin activity. This substrate has been applied to the determination of subtilisin in solution as well as for the identification of subtilisin after gel electrophoresis or isoelectrophocusing. Catalytic parameters for the new substrate were determined.     

A model substrate for solid-state fermentation

Summary A model substrate consisting of cassava starch embedded in kappa-carrageenan was used to mimic the growth ofRhizopusoligosporus on cassava tubers. Growth on the model substrate was similar to that during solid-state fermentation of the actual cassava. However, protein production and starch utilization were slower on the model substrate.     

A phylogenetically based secondary structure for the yeast telomerase RNA

BACKGROUND: Telomerase is a ribonucleoprotein complex whose RNA moiety dictates the addition of specific simple sequences onto chromosomes ends. While relevant for certain human genetic diseases, the contribution of the essential telomerase RNA to RNP assembly still remains unclear. Phylogenetic analyses of vertebrate and ciliate telomerase RNAs revealed conserved elements that potentially organize protein subunits for RNP function. In contrast, the yeast telomerase RNA could not be fitted to any known structural model, and the limited number of known sequences from Saccharomyces species did not permit the prediction of a yeast specific conserved structure. RESULTS: We cloned and analyzed the complete telomerase RNA loci (TLC1) from all known Saccharomyces species belonging to the "sensu stricto" group. Complementation analyses in S. cerevisiae and end mappings of mature RNAs ensured the relevance of the cloned sequences. By using phylogenetic comparative analysis coupled with in vitro enzymatic probing, we derived a secondary structure prediction of the Saccharomyces cerevisiae TLC1 RNA. This conserved secondary structure prediction includes a central domain that is likely to orchestrate DNA synthesis and at least two accessory domains important for RNA stability and telomerase recruitment. The structure also reveals a potential tertiary interaction between two loops in the central core. CONCLUSIONS: The predicted secondary structure of the TLC1 RNA of S. cerevisiae reveals a distinct folding pattern featuring well-separated but conserved functional elements. The predicted structure now allows for a detailed and rationally designed study to the structure-function relationships within the telomerase RNP-complex in a genetically tractable system.     

A substrate combinatorial array for caspases.

An efficient strategy for the synthesis of a tetrapeptidyl substrate combinatorial array directed toward the caspases is described. Testing of this array with caspases 1 and 4 gave substrate hydrolytic profiles characteristic of each caspase, and permitted the identification of efficiently processed substrates. A comparison of this approach to that using a positional scanning library is presented.     

A forebrain neural substrate for behavioral thermoregulation

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