A Mutant of Tetrahymena Telomerase Reverse Transcriptase with Increased Processivity

Abstract

The protein catalytic subunit of telomerase (TERT) is a reverse transcriptase (RT) that utilizes an internal RNA molecule as a template for the extension of chromosomal DNA ends. In all retroviral RTs there is a conserved tyrosine two amino acids preceding the catalytic aspartic acids in motif C, a motif that is critical for catalysis. In TERTs, however, this position is a leucine, valine, or phenylalanine. We developed and characterized a robust in vitro reconstitution system for Tetrahymena telomerase and tested the effects of amino acid substitutions on activity. Substitution of the retrovirallike tyrosine in motif C did not change overall enzymatic activity but increased processivity. This increase in processivity correlated with an increased affinity for telomeric DNA primer. Substitution of an alanine did not increase processivity, while substitution of a phenylalanine had an intermediate effect. The data suggest that this amino acid is involved in interactions with the primer in telomerase as in other RTs, and show that mutating an amino acid to that conserved in retroviral RTs makes telomerase more closely resemble these other RTs.Telomeres form a protective cap on chromosome ends and are usually composed of short G-rich DNA repeats complexed with proteins (1). The telomeres of unicellular eukaryotes and of the germ cells of multicellular organisms are maintained by the enzyme telomerase, which was first identified in the ciliated protozoan Tetrahymena thermophila (2). Telomerase activity has also been detected in many human cancer cells and appears to be necessary for their continued growth (3-5).Telomerase is a reverse transcriptase that utilizes an internal RNA molecule as a template for the extension of DNA ends (6). The RNA component of telomerase has been cloned from many different organisms (reviewed in Ref. 7). The protein catalytic subunit of telomerase, known as TERT for telomerase reverse transcriptase, was first identified in the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae (8,9) and has since been identified in humans, mice, fission yeast, plants, and other ciliated protozoa (10 -18). The evolutionary conservation of TERT 1 suggested that it and the telomerase RNA form a core catalytic unit. This was supported by experiments in which in vitro translated TERT and the telomerase RNA were sufficient to reconstitute both human and Tetrahymena telomerase activity (15,19,20), although reconstitution was dependent on the presence of chaperone proteins in the rabbit reticulocyte lysate used for in vitro translation (21, 22). The derived amino acid sequences of the TERT proteins contain motifs common to all reverse transcriptases, consistent with the role of telomerase in RNA-templated DNA polymerization (23). Within these reverse transcriptase motifs is an invariant trio of aspartic acids that are directly involved in catalysis (24,25). Mutation of any of these three aspartic acids in yeast or human TERT abolished telomerase activity in vitro and in vivo, confirming the import...

Saccharomyces cerevisiae telomerase subunit Est3p binds DNA and RNA and stimulates unwinding of RNA/DNA heteroduplexes

“…Recently, it was shown that the N-terminal domain of Tetrahymena Thermophila catalytic telomerase subunit can also bind DNA and RNA [26,27]. In human telomerase the corresponding domain [28] was shown to be crucial for telomerase processivity [29].…”

The Euplotes telomerase subunit p43 stimulates enzymatic activity and processivity in vitro

“…The stem III pseudoknot and stem-loop IV of the telomerase RNA are both required for normal levels of repeat addition processivity (Lai et al 2003;Mason et al 2003). The identity of a specific amino acid within a reverse transcriptase motif of TERT also affects repeat addition processivity (Bryan et al 2000a). Although there is generally no correlation between the mean telomere length of a given organism and the in vitro processivity of its telomerase, evidence suggests that processivity may be relevant for telomere maintenance in vivo.…”

“…Upon translation in rabbit reticulocyte lysate (RRL), Tetrahymena TERT can assemble with telomerase RNA into an active telomerase RNP (Collins and Gandhi 1998;Bryan et al 2000a) in a manner that is likely to be dependent on the action of protein chaperones (Holt et al 1999;Forsythe et al 2001). Indicative of a high degree of structural and functional complexity in telomerase, the in vitro reconstituted complex does not approach the degree of processivity of the enzyme isolated from cells (Collins and Gandhi 1998;Bryan et al 2000a).…”

“…Indicative of a high degree of structural and functional complexity in telomerase, the in vitro reconstituted complex does not approach the degree of processivity of the enzyme isolated from cells (Collins and Gandhi 1998;Bryan et al 2000a). Nevertheless, its activity resembles that of the endogenous enzyme in most other respects, making this system ideal for studying the function of telomerase accessory proteins.…”

“…Repeat addition is catalyzed by a catalytic protein subunit, the telomerase reverse transcriptase (TERT). This protein, first identified in Euplotes aediculatus, contains amino acid motifs common to all reverse transcriptases (Lingner and Cech 1996;Lingner et al 1997b) and is tethered to the RNA component via its TERT-specific N-terminal domain (Bryan et al 2000b;Lai et al 2001).…”