Nurturing interdisciplinary research.
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Biomedical Research↗
Biomedical subjects
Thomas R Cech
Publications and source records attributed to Thomas R Cech.
The Euplotes telomerase subunit p43 stimulates enzymatic activity and processivity in vitro.
Telomerase is a reverse transcriptase that synthesizes telomeric DNA repeats at the ends of eukaryotic chromosomes. Although it is minimally composed of a conserved catalytic protein subunit (TERT) and an RNA component, additional accessory factors present in the holoenzyme play crucial roles in the biogenesis and function of the enzyme complex. Telomerase from the ciliate Tetrahymena can be reconstituted in active form in vitro. Using this system, we show that p43, a telomerase-specific La-motif protein from the ciliate Euplotes, stimulates activity and increases repeat addition processivity of telomerase. Activity enhancement by p43 requires its incorporation into a TERT.RNA.p43 ternary complex but is independent of other dissociable protein factors functioning in telomerase complex assembly. Stimulation is enhanced at elevated temperatures, supporting a role for p43 in structural stabilization of a critical region of the RNA subunit. To our knowledge, this represents the first demonstration that an authentic telomerase accessory protein can directly affect the enzymatic activity of the core enzyme in vitro.
Animals↗
DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA.
Telomeres, specialized protein-DNA complexes that cap the ends of linear chromosomes, are essential for protecting chromosomes from degradation and end-to-end fusions. The Pot1 (protection of telomeres 1) protein is a widely distributed eukaryotic end-capping protein, having been identified in fission yeast, microsporidia, plants and animals. Schizosaccharomyces pombe Pot1p is essential for telomere maintenance, and human POT1 has been implicated in telomerase regulation. Pot1 binds telomeric single-stranded DNA (ssDNA) with exceptionally high sequence specificity, the molecular basis of which has been unknown. Here we describe the 1.9-A-resolution crystal structure of the amino-terminal DNA-binding domain of S. pombe Pot1p complexed with ssDNA. The protein adopts an oligonucleotide/oligosaccharide-binding (OB) fold with two loops that protrude to form a clamp for ssDNA binding. The structure explains the sequence specificity of binding: in the context of the Pot1 protein, DNA self-recognition involving base-stacking and unusual G-T base pairs compacts the DNA. Any sequence change disrupts the ability of the DNA to form this structure, preventing it from contacting the array of protein hydrogen-bonding groups. The structure also explains how Pot1p avoids binding the vast excess of RNA in the nucleus.
Base Sequence↗
Tetrahymena telomerase is active as a monomer.
Telomerase is an enzyme that utilizes an internal RNA molecule as a template for the extension of chromosomal DNA ends. The catalytic core of telomerase consists of the RNA subunit and a protein reverse transcriptase subunit, known as telomerase reverse transcriptase (TERT). It has previously been shown that both yeast and human telomerase can form dimers or multimers in which one RNA in the complex can influence the activity of another. To test the proposal that dimerization might be essential for telomerase activity, we sought to determine whether Tetrahymena thermophila telomerase is active as a dimer or a monomer. Recombinant Tetrahymena telomerase eluted from a gel filtration column at the size of a monomeric complex (one RNA plus one TERT), and those fractions showed processive telomerase activity. We were unable to detect dimerization of Tetrahymena telomerase by coprecipitation experiments, by using tags on either the TERT protein or telomerase RNA. Therefore, a majority, if not all, of the recombinant Tetrahymena telomerase in our reconstitution system is present as a monomeric complex. We were also unable to detect dimerization of native telomerase from mating and vegetative Tetrahymena cell extracts. These results demonstrate that Tetrahymena telomerase does not need to dimerize to be active and processive.
Animals↗
Sequence-specific and 3'-end selective single-strand DNA binding by the Oxytricha nova telomere end binding protein alpha subunit.
Oxytricha nova telomere end binding protein (OnTEBP) specifically recognizes and caps single-strand (T(4)G(4))(2) telomeric DNA at the very 3'-ends of O. nova macronuclear chromosomes. The discovery of proteins homologous to the N-terminal domain of the OnTEBP alpha subunit in Euplotes crassus, Schizosaccharomyces pombe, and Homo sapiens suggests that related proteins are widely distributed in eukaryotes. Previously reported crystal structures of the ssDNA binding domain of the OnTEBP alpha subunit both uncomplexed and complexed with telomeric ssDNA have suggested specific mechanisms for sequence-specific and 3'-end selective recognition of the single-strand telomeric DNA. We now describe comparative binding studies of ssDNA recognition by the N-terminal domain of the OnTEBP alpha subunit. Addition of nucleotides to the 3'-end of the TTTTGGGG telomere repeat decreases the level of alpha binding by up to 7-fold, revealing a modest specificity for a 3'-terminus relative to an internal DNA binding site. Nucleotide substitutions at specific positions within the t(1)t(2)t(3)T(4)G(5)G(6)G(7)G(8) repeat show that base substitutions at some sites do not substantially decrease the binding affinity (<2-fold for lowercase letters), while substitutions at other sites dramatically reduce the binding affinity (>20-fold decrease for the uppercase bold letter). Comparison of the structural and binding data provides unique insights into the ways in which proteins recognize and bind single-stranded DNA.
Animals↗
Human POT1 facilitates telomere elongation by telomerase.
Mammalian telomeric DNA is mostly composed of double-stranded 5'-TTAGGG-3' repeats and ends with a single-stranded 3' overhang. Telomeric proteins stabilize the telomere by protecting the overhang from degradation or by remodeling the telomere into a T loop structure. Telomerase is a ribonucleoprotein that synthesizes new telomeric DNA. In budding yeast, other proteins, such as Cdc13p, that may help maintain the telomere end by regulating the recruitment or local activity of telomerase have been identified. Pot1 is a single-stranded telomeric DNA binding protein first identified in fission yeast, where it was shown to protect telomeres from degradation [10]. Human POT1 (hPOT1) protein is known to bind specifically to the G-rich telomere strand. We now show that hPOT1 can act as a telomerase-dependent, positive regulator of telomere length. Three splice variants of hPOT1 were overexpressed in a telomerase-positive human cell line. All three variants lengthened telomeres, and splice variant 1 was the most effective. hPOT1 was unable to lengthen the telomeres of telomerase-negative cells unless telomerase activity was induced. These data suggest that a normal function of hPOT1 is to facilitate telomere elongation by telomerase.
Cell Line↗
The Euplotes La motif protein p43 has properties of a telomerase-specific subunit.
Telomerase is a specialized reverse transcriptase synthesizing DNA repeats at telomeres. In addition to the RNA and catalytic protein components, telomerase from the ciliate Euplotes aediculatus contains the subunit p43. This protein is homologous to the La autoantigen, functioning in maturation of RNA polymerase III transcripts. Here we provide evidence that p43 is primarily associated with the telomerase ribonucleoprotein in vivo. Recombinant p43 binds telomerase RNA with low-nanomolar affinity in vitro, recognizing stem I and adjacent nucleotides or structures in the core of the RNA. Unlike authentic La proteins, p43 does not bind strongly to RNA polymerase III precursor transcripts and does not exhibit a marked binding preference for 3'-terminal oligouridylate residues. In isolated macronuclei, p43 largely colocalizes with telomerase RNA in discrete foci. These findings suggest that p43 is not the Euplotes La protein but instead plays a dedicated role in telomerase assembly and/or function. Thus, p43 joins the telomerase reverse transcriptase and the yeast proteins Est1p and Est3p as the only telomerase-specific proteins identified so far.
Animals↗
Yeast telomerase is specialized for C/A-rich RNA templates.
Telomeres, the protective caps of eukaryotic chromosomes, are maintained by the enzyme telomerase. This telomere-specific reverse transcriptase (RT) uses a small region of its RNA subunit as template to synthesize telomeric DNA, which is generally G/T rich in the strand that contains the 3' end. To further our understanding of why telomeres are usually G/T rich, we screened Saccharomyces cerevisiae telomerase RNA (TLC1) libraries with randomized template sequences for complementation of a tlc1 deletion and decapping of existing telomeres. Surprisingly, the vast majority of the 60 000 different mutant telomerase templates tested showed no activity in vivo. This deficiency was not due to impaired assembly with the catalytic subunit (Est2p) nor could it be alleviated by enforced telomerase recruitment to the telomeres. Rather, the mutant templates reduced the nucleotide addition processivity of telomerase. The functional RNA template sequences recovered in our screens preferentially contained two or more consecutive rC nucleotides, reminiscent of the wild-type template. Thus, in contrast to retroviral RTs that can reverse transcribe any RNA sequence into DNA, the budding yeast telomerase RT is specialized for its C-rich RNA template.
Adenine↗
Rebalancing teaching and research.
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Academies and Institutes↗
N-terminal domain of yeast telomerase reverse transcriptase: recruitment of Est3p to the telomerase complex.
Telomerase is a reverse transcriptase that maintains chromosome ends. The N-terminal half of the catalytic protein subunit (TERT) contains three functional domains (I, II, and III) that are conserved among TERTs but not found in other reverse transcriptases. Guided by an amino acid sequence alignment of nine TERT proteins, mutations were introduced into yeast TERT (Est2p). In support of the proposed alignment, mutation of virtually all conserved residues resulted in loss-of-function or temperature sensitivity, accompanied by telomere shortening. Overexpression of telomerase component Est3p led to allele-specific suppression of the temperature-sensitive mutations in region I, suggesting that Est3p interacts with this protein domain. As predicted by the genetic results, a lethal mutation in region I resulted in loss of Est3p from the telomerase complex. We conclude that Est2p region I is required for the recruitment of Est3p to yeast telomerase. Given the phylogenetic conservation of region I of TERT, this protein domain may provide the equivalent function in all telomerases.
Amino Acid Sequence↗
Sharing publication-related data and materials: responsibilities of authorship in the life sciences.
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Authorship↗
A template-proximal RNA paired element contributes to Saccharomyces cerevisiae telomerase activity.
The ribonucleoprotein complex telomerase is critical for replenishing chromosome-end sequence during eukaryotic DNA replication. The template for the addition of telomeric repeats is provided by the RNA component of telomerase. However, in budding yeast, little is known about the structure and function of most of the remainder of the telomerase RNA. Here, we report the identification of a paired element located immediately 5' of the template region in the Saccharomyces cerevisiae telomerase RNA. Mutations disrupting or replacing the helical element showed that this structure, but not its exact nucleotide sequence, is important for telomerase function in vivo and in vitro. Biochemical characterization of a paired element mutant showed that the mutant generated longer products and incorporated noncognate nucleotides. Sequencing of in vivo synthesized telomeres from this mutant showed that DNA synthesis proceeded beyond the normal template. Thus, the S. cerevisiae element resembles a similar element found in Kluyveromyces budding yeasts with respect to a function in template boundary specification. In addition, the in vitro activity of the paired element mutant indicates that the RNA element has additional functions in enzyme processivity and in directing template usage by telomerase.
Base Sequence↗
Cooperative binding of single-stranded telomeric DNA by the Pot1 protein of Schizosaccharomyces pombe.
The fission yeast Pot1 (protection of telomeres) protein is a single-stranded telomeric DNA-binding protein and is required to protect the ends of chromosomes. Its N-terminal DNA-binding domain, Pot1pN, shows sequence similarity to the first OB fold of the telomere-binding protein alpha subunit of Oxytricha nova. The minimal-length telomeric ssDNA required to bind Pot1pN was determined to consist of six nucleotides, GGTTAC, by gel filtration chromatography and filter-binding assay (K(D) = 83 nM). Pot1pN is a monomer, and each monomer binds one hexanucleotide. Experiments with nucleotide substitutions demonstrated that the central four nucleotides are crucial for binding. The dependence of Pot1pN-ssDNA binding on salt concentration was consistent with a single ionic contact between the protein and the ssDNA phosphate backbone, such that at physiological salt condition 83% of the free energy of binding is nonelectrostatic. Subsequent binding experiments with longer ssDNAs indicated that Pot1pN binds to telomeric ssDNA with 3' end preference and in a highly cooperative manner that mainly results from DNA-induced protein-protein interactions. Together, the binding properties of Pot1pN suggest that the protein anchors itself at the very 3' end of a chromosome and then fills in very efficiently, coating the entire single-stranded overhang of the telomere.
Animals↗
A bulged stem tethers Est1p to telomerase RNA in budding yeast.
It is well established that the template for telomeric DNA synthesis is provided by the RNA subunit of telomerase; however, the additional functions provided by most of the rest of the RNA (>1000 nucleotides in budding yeast) are largely unknown. By alignment of telomerase RNAs of Saccharomyces cerevisiae and six Kluyveromyces species followed by mutagenesis of the S. cerevisiae RNA, we found a conserved region that is essential for telomere maintenance. Phylogenetic analysis and computer folding revealed that this region is conserved not only in primary nucleotide sequence but also in secondary structure. A common bulged-stem structure was predicted in all seven yeast species. Mutational analysis showed the structure to be essential for telomerase function. Suppression of bulged-stem mutant phenotypes by overexpression of Est1p and loss of co-immunoprecipitation of the mutant RNAs with Est1p indicated that this bulged stem is necessary for association of Est1p, a telomerase regulatory subunit. Est1p in yeast extract bound specifically to a small RNA containing the bulged stem, suggesting a direct interaction. We propose that this RNA structure links the enzymatic core of telomerase with Est1p, thereby allowing Est1p to recruit or activate telomerase at the telomere.
Conserved Sequence↗
The chemical repertoire of natural ribozymes.
Although RNA is generally thought to be a passive genetic blueprint, some RNA molecules, called ribozymes, have intrinsic enzyme-like activity--they can catalyse chemical reactions in the complete absence of protein cofactors. In addition to the well-known small ribozymes that cleave phosphodiester bonds, we now know that RNA catalysts probably effect a number of key cellular reactions. This versatility has lent credence to the idea that RNA molecules may have been central to the early stages of life on Earth.
Animals↗
Effects of nucleotide analogues on Euplotes aediculatus telomerase processivity: evidence for product-assisted translocation.
Telomerase is a unique ribonucleoprotein that reverse transcribes a defined region of its RNA subunit onto the ends of eukaryotic chromosomes. The product of telomerase, telomeric DNA, is typically a G-rich repeated sequence, (TTTTGGGG)(n) in the ciliate Euplotes aediculatus and (TTAGGG)(n) in humans. Telomerase can extend oligonucleotide primers in vitro in a processive fashion. We used dNTP analogues to study the structure-activity relationship between substrate nucleotides and processivity of telomerase from E. aediculatus. Several analogues, including 2'-deoxyuridine triphosphate (dUTP), 2'-deoxyinosine triphosphate (dITP), and 7-deaza-2'-deoxyguanosine triphosphate (7-deaza-dGTP), were good substrates for telomerase with K(m) and V(max) values near those of the natural substrates, dTTP and dGTP. However, telomerase processivity was affected with these substrates, decreasing in the order dUTP > 7-deaza-dGTP > dITP. Telomerase did not completely reverse transcribe the template when dITP was the substrate, and it efficiently extended a primer by the addition of two repeats when 7-deaza-dGTP and dUTP were utilized. When the same nucleotide analogues were incorporated into the primers, no effects were observed except in the case of a 3'-terminal deoxyinosine. The data support a model that includes the formation of an intramolecular secondary structure within the product DNA to facilitate translocation. The most likely structure is a G-G hairpin.
Animals↗
HHMI's attention to Wiley's lab staff.
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Academies and Institutes↗
In vivo selection of better self-splicing introns in Escherichia coli: the role of the P1 extension helix of the Tetrahymena intron.
In vivo selection was used to improve the activity of the Tetrahymena pre-rRNA self-splicing intron in the context of heterologous exons. The intron was engineered into a kanamycin nucleotidyltransferase gene, with the pairing between intron bases and the 5' and 3' splice sites maintained. The initial construct failed to confer kanamycin resistance on Escherichia coli, although the pre-mRNA was active in splicing in vitro. Random mutation libraries were constructed to identify active intron variants in E. coli. All the active mutants sequenced contained mutations disrupting a base-paired region above the paired region P1 (referred to as the P1 extension region or P1ex) that involves the very 5' end of the intron. Subsequent site-directed mutagenesis confirmed that these P1ex mutations are responsible and sufficient to activate the intron splicing in E. coli. Thus, it appears that too strong of a secondary structure in the P1ex element can be inhibitory to splicing in vivo. In vitro splicing assays demonstrated that two P1ex mutant constructs splice six to eight times faster than the designed construct at 40 microM GTP concentration. The relative reaction rates of the mutant constructs compared to the original design are further increased at a lower GTP concentration. Possible mechanisms by which the disrupted P1ex structure could influence splicing rates are discussed. This study emphasizes the value of using libraries of random mutations to improve the activity of ribozymes in heterologous contexts in vivo.
Animals↗