scholarly journals Identification of Kluyveromyces lactisTelomerase: Discontinuous Synthesis along the 30-Nucleotide-Long Templating Domain

1998 ◽  
Vol 18 (9) ◽  
pp. 4961-4970 ◽  
Author(s):  
Tracy Boswell Fulton ◽  
Elizabeth H. Blackburn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kluyveromyces Lactis ◽  
Telomerase Activity ◽  
Reaction Products ◽  
Telomeric Dna ◽  
Whole Cell ◽  
Cell Extracts ◽  
Dna Template ◽  
Insight Into

ABSTRACT Telomeres in the budding yeast Kluyveromyces lactisconsist of perfectly repeated 25-bp units, unlike the imprecise repeats at Saccharomyces cerevisiae telomeres and the short (6- to 8-bp) telomeric repeats found in many other eukaryotes. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which uses a portion of its RNA moiety as a template. K. lactistelomerase RNA, encoded by the TER1 gene, is ∼1.3 kb long and contains a 30-nucleotide templating domain, the largest ever examined. To examine the mechanism of polymerization by this enzyme, we identified and analyzed telomerase activity from K. lactiswhole-cell extracts. In this study, we exploited the length of the template and the precision of copying by K. lactistelomerase to examine primer elongation within one round of repeat synthesis. Under all in vitro conditions tested, K. lactistelomerase catalyzed only one round of repeat synthesis and remained bound to reaction products. We demonstrate that K. lactistelomerase polymerizes along the template in a discontinuous manner and stalls at two specific regions in the template. Increasing the amount of primer DNA-template RNA complementarity results in stalling, suggesting that the RNA-DNA hybrid is not unpaired during elongation in vitro and that lengthy duplexes hinder polymerization through particular regions of the template. We suggest that these observations provide an insight into the mechanism of telomerase and its regulation.

scholarly journals Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

2008 ◽  
Vol 28 (7) ◽  
pp. 2380-2390 ◽  
Author(s):  
Hong Ji ◽  
Christopher J. Adkins ◽  
Bethany R. Cartwright ◽  
Katherine L. Friedman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Specific Binding ◽  
Telomerase Activity ◽  
Negative Regulator ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomere Length Homeostasis

ABSTRACT In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.

scholarly journals Plasmodium falciparum Telomerase: De Novo Telomere Addition to Telomeric and Nontelomeric Sequences and Role in Chromosome Healing

1998 ◽  
Vol 18 (2) ◽  
pp. 919-925 ◽  
Author(s):  
Emmanuel Bottius ◽  
Nassera Bakhsis ◽  
Artur Scherf
Keyword(s):  
Plasmodium Falciparum ◽  
Reverse Transcriptase ◽  
De Novo ◽  
Telomerase Activity ◽  
Telomeric Dna ◽  
Oligonucleotide Primers ◽  
Cell Extracts ◽  
Linear Chromosomes ◽  
Dna Primers

ABSTRACT Telomerase, a specialized cellular reverse transcriptase, compensates for chromosome shortening during the proliferation of most eucaryotic cells and contributes to cellular immortalization. The mechanism used by the single-celled protozoan malaria parasitePlasmodium falciparum to complete the replication of its linear chromosomes is currently unknown. In this study, telomerase activity has for the first time been identified in cell extracts ofP. falciparum. The de novo synthesis of highly variable telomere repeats to the 3′ end of DNA oligonucleotide primers by plasmodial telomerase is demonstrated. Permutated telomeric DNA primers are extended by the addition of the next correct base. In addition to elongating preexisting telomere sequences, P. falciparumtelomerase can also add telomere repeats onto nontelomeric 3′ ends. The sequence GGGTT… was the predominant initial DNA sequence added to the nontelomeric 3′ ends in vitro. Poly(C) at the 3′ end of the oligonucleotide significantly alters the precision of the new telomerase added repeats. The efficiency of nontelomeric primer elongation was dependent on the presence of a G-rich cassette upstream of the 3′ terminus. Oligonucleotide primers based on natural P. falciparum chromosome breakpoints are efficiently used as telomerase substrates. These results imply that P. falciparum telomerase contributes to chromosome maintenance and to de novo telomere formation on broken chromosomes. Reverse transcriptase inhibitors such as dideoxy GTP efficiently inhibitP. falciparum telomerase activity in vitro. These data point to malaria telomerase as a new target for the development of drugs that could induce parasite cell senescence.

Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase

1986 ◽  
Vol 6 (7) ◽  
pp. 2382-2391
Author(s):  
C A Kaiser ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Mutant Gene ◽  
Yeast Cells ◽  
Molecular Weight Characteristic ◽  
Cell Extracts ◽  
Substitution Mutations ◽  
Membrane Components

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

scholarly journals ADR1-Mediated Transcriptional Activation Requires the Presence of an Intact TFIID Complex

1998 ◽  
Vol 18 (10) ◽  
pp. 5861-5867 ◽  
Author(s):  
Philip B. Komarnitsky ◽  
Edward R. Klebanow ◽  
P. Anthony Weil ◽  
Clyde L. Denis
Keyword(s):  
Transcriptional Activation ◽  
Yeast Cells ◽  
Binding Studies ◽  
Whole Cell ◽  
Transactivation Domains ◽  
Cell Extracts ◽  
Genes Expression ◽  
Tfiid Complex

ABSTRACT The yeast transcriptional activator ADR1, which is required forADH2 and other genes’ expression, contains four transactivation domains (TADs). While previous studies have shown that these TADs act through GCN5 and ADA2, and presumably TFIIB, other factors are likely to be involved in ADR1 function. In this study, we addressed the question of whether TFIID is also required for ADR1 action. In vitro binding studies indicated that TADI of ADR1 was able to retain TAFII90 from yeast extracts and TADII could retain TBP and TAFII130/145. TADIV, however, was capable of retaining multiple TAFIIs, suggesting that TADIV was binding TFIID from yeast whole-cell extracts. The ability of TADIV truncation derivatives to interact with TFIID correlated with their transcription activation potential in vivo. In addition, the ability of LexA-ADR1-TADIV to activate transcription in vivo was compromised by a mutation in TAFII130/145. ADR1 was found to associate in vivo with TFIID in that immunoprecipitation of either TAFII90 or TBP from yeast whole-cell extracts specifically coimmunoprecipitated ADR1. Most importantly, depletion of TAFII90 from yeast cells dramatically reducedADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex.

Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae

1991 ◽  
Vol 11 (9) ◽  
pp. 4555-4560 ◽  
Author(s):  
M Woontner ◽  
P A Wade ◽  
J Bonner ◽  
J A Jaehning
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
In Vitro Transcription ◽  
Cell Extract ◽  
Whole Cell ◽  
Transcription System ◽  
Upstream Activation Sequence ◽  
Activation Sequence

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.

scholarly journals The conserved structure of plant telomerase RNA provides the missing link for an evolutionary pathway from ciliates to humans

2019 ◽  
Vol 116 (49) ◽  
pp. 24542-24550 ◽  
Author(s):  
Jiarui Song ◽  
Dhenugen Logeswaran ◽  
Claudia Castillo-González ◽  
Yang Li ◽  
Sreyashree Bose ◽  
...  
Keyword(s):  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Structural Element ◽  
Stem Loop ◽  
Evolutionary Pathway ◽  
Pol Iii

Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3′ long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.

scholarly journals Towards structural characterization of H. polymorpha telomerase components

2014 ◽  
Vol 70 (a1) ◽  
pp. C816-C816
Author(s):  
Johanna Kallio ◽  
Elena Rodina ◽  
Maria Zvereva ◽  
Olga Petrova ◽  
Alexandr Malyavko ◽  
...  
Keyword(s):  
Cell Division ◽  
Structural Information ◽  
Hansenula Polymorpha ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Premature Aging ◽  
Telomeric Dna ◽  
Terminal Domain ◽  
Protein Components

Telomeres are regions of non-coding DNA that cap the chromosomes, preventing the loss of coding DNA during cell division and contributing to chromosomal stability. In actively dividing cells, such as embryonic stem cells, the telomeres need to elongated by telomerase. The telomerase complex consist of the enzyme telomerase reverse transcriptase (TERT), telomerase RNA (TR) and additional proteins. TERT and TR are required for the telomerase activity in vitro. Telomerase is active in vast majority of the cancer cells ensuring continuous cell division and tumor growth. Syndromes leading to premature aging are often associated with short telomeres. Finding ways to regulate the telomerase activity would help to advance therapies for these conditions. However, the structural information available of the telomerase complex is very limited. We have chosen thermophilic yeast Hansenula polymorpha as a model system due to the stability of its proteins. The N-terminal domain of the TERT is essential for telomerase activity and possibly is involved in binding of TR, telomeric DNA and additional protein components of the telomerase complex. We have crystallised the N-terminal domain of H. polymorpha TERT and, in lack of a homologious structure, produced a seleno-methionine derivative of the protein. MAD data on N-terminal domain has been collected to resolution of 2.0 Å at the PETRA-III beamline P13 (EMBL/DESY) in Hamburg. We will discuss the structure-function relationship of the N-domain and the whole TERT component.

scholarly journals Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase.

1986 ◽  
Vol 6 (7) ◽  
pp. 2382-2391 ◽  
Author(s):  
C A Kaiser ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Mutant Gene ◽  
Yeast Cells ◽  
Molecular Weight Characteristic ◽  
Cell Extracts ◽  
Substitution Mutations ◽  
Membrane Components

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

scholarly journals Two RNA subunits and POT1a are components of Arabidopsis telomerase

2010 ◽  
Vol 108 (1) ◽  
pp. 73-78 ◽  
Author(s):  
Catherine Cifuentes-Rojas ◽  
Kalpana Kannan ◽  
Lin Tseng ◽  
Dorothy E. Shippen
Keyword(s):  
Telomerase Activity ◽  
Flowering Plant ◽  
Telomeric Dna ◽  
Telomere Shortening ◽  
Telomere Repeat ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions ◽  
Novel Protein

Telomerase is a ribonucleoprotein (RNP) reverse transcriptase whose essential RNA subunit (TER) functions as a template for telomere repeat synthesis. Here we report the identification of two divergent TER moieties in the flowering plant Arabidopsis thaliana. Although both TER1 and TER2 copurify with telomerase activity and serve as templates for telomerase in vitro, depletion of TER1, but not TER2, leads to decreased telomerase activity and progressive telomere shortening in vivo. Moreover, mutation of the templating domain in TER1 results in the incorporation of mutant telomere repeats on chromosome ends. Thus, TER1 provides the major template for telomerase in vivo. We also show that POT1a binds TER1 with a Kd of 2 × 10-7 M and the two components assemble into an enzymatically active RNP in vivo. In contrast, TER1-POT1b and TER2-POT1a associations were not observed. In other organisms POT1 proteins bind telomeric DNA and provide chromosome end protection. We propose that duplication of TER and POT1 in Arabidopsis fueled the evolution of novel protein–nucleic acid interactions and the migration of POT1 from the telomere to the telomerase RNP.

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