The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation. In addition to the human telomerase RNA component (hTR; ref. 11), TR1/TLP1 (refs 12, 13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (5pTrt1) telomerases, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans. Activation of telomerase is often associated with immortalization of human cells and cancer. To dissect the human telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential 445 nucleotide human telomerase RNA (hTR) by micrococcal nuclease digestion of partially purified human telomerase, the addition of in vitro transcribed hTR reconstituted telomerase activity. The activity was dependent upon and specific to hTR. Using this assay, truncations at the 5′ and 3′ ends of hTR identified a functional region of hTR, similar in size to the full‐length telomerase RNAs from ciliates. This region is located between positions 1‐203. Furthermore, we found that residues 1‐44, 5′ to the template region (residues 46–56) are not essential for activity, indicating a minimal functional region is located between residues 44–203. Mutagenesis of full‐length hTR between residues 170–179, 180–189 or 190–199 almost completely abolished the ability of the hTR to function in the reconstitution of telomerase activity, suggesting that sequences or structures within this 30 nucleotide region are required for activity, perhaps by binding telomerase protein components.

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.

We have designed, synthesized, and evaluated using physical, chemical and biochemical assays various oligonucleotide N3'-->P5' phosphoramidates, as potential telomerase inhibitors. Among the prepared compounds were 2'-deoxy, 2'-hydroxy, 2'-methoxy, 2'-ribo-fluoro, and 2'-arabino-fluoro oligonucleotide phosphoramidates, as well as novel N3'-->P5' thio-phosphoramidates. The compounds demonstrated sequence specific and dose dependent activity with IC50 values in the sub-nM to pM concentration range.

The early E2 (E2E) promoter of adenovirus type 2 possesses a TATA-like element and binding sites for the factors E2F and ATF. This promoter is transcribed by RNA polymerase II in high salt nuclear extracts, but by RNA polymerase III in standard nuclear extracts, as judged by sensitivity to low and high, respectively, concentrations of alpha-amanitin. Transcription by the two RNA polymerases initiated at the same site and depended, in both cases, on the TATA-like sequence and upstream elements. However, RNA polymerase III transcripts, unlike those synthesized by RNA polymerase II, terminated at two runs of Ts downstream of the initiation site. Although they are not essential, sequences downstream of the initiation site increased the efficiency of E2E transcription by RNA polymerase III. Such RNA polymerase III dependent transcription required a subpopulation of the general transcription factor, TFIID: TFIID that binds weakly to phosphocellulose (0.3 M eluate) complemented a TFIID-depleted extract to restore RNAp III transcription, whereas TFIID tightly associated with phosphocellulose (1 M eluate) was unable to do so.

We have previously reported that the subgroup C adenovirus E2 early (E2E) RNA polymerase 11 promoter can specify efficient in vitro transcription by RNA polymerase m. We now show that promoter proximal sequences of the E2E transcription unit are also transcribed by RNA polymerase Ill in nuclei isolated from adenovirusinfected cells. Small E2E RNA species that possessed the same properties as in vitro synthesized RNA polymerase m E2E transcripts were detected in cytoplasmic RNA populations from infected cells by using blotting, primer extension, and RNase protection assays. The 3' termini of these RNAs were mapped to thymidine-rich sequences typical of RNA polymerase m termination sites. These results demonstrate that a single gene can be transcribed by both RNA polymerase II and RNA polymerase m in vivo.The ubiquitous function of the TATA-binding protein (TBP; see ref. 1) in initiation of eukaryotic transcription (refs. 2 and 3; see also refs. 4 and 5) suggests that the three eukaryotic RNA polymerases employ fundamentally similar mechanisms of initiation (4, 5). Certain RNA polymerase (pol) III promoters, moreover, closely resemble typical pol II promoters (6-8): for example, vertebrate U6 promoters comprise a set of upstream elements, including a TATA sequence essential for recognition by pol III (6-8). These properties suggest that some promoters might be transcribed by both enzymes. Although pol II transcription from the Xenopus U6 promoter and pol III transcription from several pol II promoters (9-12, 27) have been observed in experimental situations, such dual transcription has not been demonstrated in vivo. A pol III promoter which shares upstream elements with the well-characterized adenovirus type 2 (Ad2) E2E pol II promoter, is active in vitro (11). The comparable in vitro activities of the pol II and pol III E2E promoters prompted us to search adenovirus-infected cells for analogues of products of in vitro pol III transcription (Fig. 1 of the coding, third exon (see Fig. 1A) were synthesized, purified, and 5' end-labeled as described (11).Run-On Transcription Assays. RNA labeled in run-on transcription mixtures containing 108 nuclei from Ad2-infected cells was purified and hybridized (15) to dot blots carrying the E2E -17 to + 120 antisense RNA (2 Zg per dot) or linearized plasmids containing either the Ad2 major late (ML) L2 plus tripartite leader sequences (2.5 jig per dot) or the viral VA-RNA, gene (2.5 ,ug per dot). Membranes were exposed for autoradiography, or the hybridized RNA was eluted by heating at 950C for 10 min in 5 mM EDTA prior to electrophoresis in 6% polyacrylamide sequencing gels (16).In Vitro Transcription. pol III transcripts of the Ad2 E2E promoter were synthesized in the presence of a-amanitin (2 pg/ml) (17).Preparation and Analysis of RNA from Ad2-Infected Cells.Cytoplasmic RNA was purified from uninfected or Ad2-infected HeLa cells (18) and separated into poly(A)-containing and poly(A)-lacking populations (17), or into populations precipitable or soluble in 2.5 M LiC...

Telomerase is a ribonucleoprotein responsible for maintaining telomeres in nearly all eukaryotic cells. The enzyme is able to utilize a short segment of its RNA subunit as the template for the reverse transcription of d(TTAGGG) repeats onto the ends of human chromosomes. Transfection with telomerase was shown to confer immortality on several types of human cells. Moreover, telomerase activation appears to be one of the key events required for malignant transformation of normal cells. Inhibition of telomerase activity in transformed cells results in the cessation of cell proliferation in cultures and provides the rationale for the selection of telomerase as a target for anticancer therapy. Using oligonucleotide N3'-->P5' phosphoramidates (NPs) we have identified a region of the human telomerase RNA subunit (hTR) approximately 100 nt downstream from the template region whose structural integrity appears crucial for telomerase enzymatic activity. The oligonucleotides targeted to this segment of hTR are potent and specific inhibitors of telomerase activity in biochemical assays. Mutant telomerase, in which 3 nt of hTR were not complementary to a 15 nt NP, was found to be refractory to inhibition by that oligonucleotide. We also demonstrated that the binding of NP, oligonucleotides to this hTR allosteric site results in a marked decrease in the affinity of a telomerase substrate (single-stranded DNA primer) for the enzyme.

Nuclear extracts and viral transcribing minichromosomes were prepared from SV40‐infected cells and incubated in vitro with [alpha‐32P]UTP under conditions which allow the elongation of preinitiated RNA chains. Sucrose gradient lysis of the transcription mixtures revealed two populations of SV40‐specific RNA: elongating chains that remain associated with the viral minichromosomes, and, at the top of the gradient, small free RNA detached from the template and hybridizing exclusively to the promoter‐proximal region of SV40 DNA. This free RNA was shown by polyacrylamide gel electrophoresis to comprise essentially a 94 nucleotide species, which could, however, at high UTP concentration, be elongated a further few nucleotides before terminating. These results thus show that the actively transcribing minichromosomes provide a sytem in which the attenuated RNA can be released from the template. Moreover, this is the first demonstration of specific in vitro termination of polymerase B transcription. The conditions which lead to transcription termination are discussed.