Identification of an essential proximal sequence element in the promoter of the telomerase RNA gene of Tetrahymena thermophila

Hargrove, Brian W.; Bhattacharyya, Anamitra; Domitrovich, Angela M.; Kapler, Geoffrey M.; Kirk, Karen E.; Shippen, Dorothy E.; Kunkel, Gary R.

doi:10.1093/nar/27.21.4269

Cited by 16 publications

(13 citation statements)

References 38 publications

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“…Although TATA and CG boxes seem to be absent in Tetrahymena promoters (7), a few regulatory elements have been identified by deletion and mutational analysis. For example, a PSE element was found to be conserved in the promoters of ciliate telomerase genes, and three elements were identified in the promoter of the RAD-51 gene (17,35). Among the latter, only the UV1-UV2 repeats are homologous to the damage-responsive elements of yeasts (20).…”

Section: Discussionmentioning

confidence: 99%

Combination of Two Regulatory Elements in the Tetrahymena thermophila HSP70-1 Gene Controls Heat Shock Activation

et al. 2008

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The induction of heat shock genes (HSPs) is thought to be primarily regulated by heat shock transcription factors (HSFs), which bind target sequences on HSP promoters, called heat shock elements (HSEs). In this study, we investigated the 5 untranslated regions of the Tetrahymena thermophila HSP70-1 gene, and we found, in addition to the canonical and divergent HSEs, multiple sets of GATA elements that have not been reported previously in protozoa. By means of in vivo analysis of a green fluorescent protein reporter transgene driven by the HSP70-1 promoter, we demonstrate that HSEs do not represent the minimal regulatory elements for heat shock induction, since the HSP70-1 is tightly regulated by both HSE and GATA elements. Electrophoretic mobility shift assay also showed that HSFs are constitutively bound to the HSEs, whereas GATA elements are engaged only after heat shock. This is the first demonstration by in vivo analysis of functional HSE and GATA elements in protozoa. Furthermore, we provide evidence of a functional link between HSE and GATA elements in the activation of the heat shock response.The heat shock response is a universal property organisms use to cope with injuries caused by elevated temperatures, infections, exposure to toxic elements, and other environmental stresses. It has been established as a paradigm for inducible gene expression, leading researchers to unravel fundamental questions related to the molecular mechanisms of gene switches. The heat shock response involves a rapid and massive synthesis of a set of the so-called heat shock proteins (HSPs), among which those with a molecular mass of ϳ70 KDa (HSP70s) play key roles (18).The stress induction of the HSP70s is generally regulated at the transcriptional level by the binding heat shock transcription factors (HSFs) to the heat shock elements (HSEs) present in the 5Ј flanking regions of heat shock genes. HSEs consist of contiguous inverted repeats of the sequence motif 5Ј-nGAAn3-Ј. The functional element usually contains a minimum of three repeated motifs with a maximum insertion of 5 bp between each motif (29). Nevertheless, functional HSEs may also digress from the consensus sequences. Among the deviations, the so-called gapped HSEs are frequently observed, containing a number of bases inserted between the repetitions. The Saccharomyces cerevisiae MDJ1 promoter is an example of a functional gapped HSE since it carries an insertion of 11 bp between the first and the second repetition [nTTCn-(11 bp)-nGAAn-(5 bp)-nGAAn]. This promoter also contains two functional contiguous nGAAn direct repeats (38). Recently, several examples of functional HSEs with direct repeats of nTTCn or nGAAn interrupted by 5-bp insertions were characterized in the promoter regions of several S. cerevisiae genes (34, 41).Ciliated protozoa are unicellular eukaryotic organisms that are exposed to the natural environment for their entire life cycle. As a result, they serve as ideal organisms for studying molecular responses to the many stressful environmental conditi...

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Section: Discussionmentioning

confidence: 99%

Combination of Two Regulatory Elements in the Tetrahymena thermophila HSP70-1 Gene Controls Heat Shock Activation

et al. 2008

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“…Several observations favor the idea that 26T RNA is processed from an rRNA precursor. The DNA segment upstream of the 26T interval lacks sequence motifs expected for transcription from an internal RNA polymerase I, II or III promoter (Gallagher and Blackburn, 1998; Hargrove et al , 1999). The primary sequence and RNA secondary structure of this region are conserved, suggesting that it is constrained by its role in the ribosome.…”

Section: Discussionmentioning

confidence: 99%

Tetrahymena ORC contains a ribosomal RNA fragment that participates in rDNA origin recognition

Mohammad

Donti

Yakisich

et al. 2007

EMBO J

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The Tetrahymena thermophila ribosomal DNA (rDNA) replicon contains dispersed cis-acting replication determinants, including reiterated type I elements that associate with sequence-specific, single-stranded binding factors, TIF1 through TIF4. Here, we show that TIF4, previously implicated in cell cycle-controlled DNA replication and rDNA gene amplification, is the T. thermophila origin recognition complex (TtORC). We further demonstrate that TtORC contains an integral RNA subunit that participates in rDNA origin recognition. Remarkably, this RNA, designated 26T, spans the terminal 282 nts of 26S ribosomal RNA. 26T RNA exhibits extensive complementarity to the type I element T-rich strand and binds the rDNA origin in vivo. Mutations that disrupt predicted interactions between 26T RNA and its complementary rDNA target change the in vitro binding specificity of ORC and diminish in vivo rDNA origin utilization. These findings reveal a role for ribosomal RNA in chromosome biology and define a new mechanism for targeting ORC to replication initiation sites.

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“…The stem II tag location replaced position 28; the 3Ј tag location was after position 155. Recombinant hairpin-tagged TER (hpTER) was expressed from a transgene containing TER sequence and 150 bp upstream, including the TER promoter (17). Transcription termination was enforced with a T 7 tract in the transcript strand.…”

Section: Methodsmentioning

confidence: 99%

Biological and Biochemical Functions of RNA in the Tetrahymena Telomerase Holoenzyme

Cunningham

Collins

2005

Molecular and Cellular Biology

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Telomerase extends chromosome ends by the synthesis of tandem simple-sequence repeats. Studies of minimal recombinant telomerase ribonucleoprotein (RNP) reconstituted in vitro have revealed sequences within the telomerase RNA subunit (TER) that are required to establish its internal template and other unique features of enzyme activity. Here we test the significance of these motifs following TER assembly into telomerase holoenzyme in vivo. We established a method for stable expression of epitope-tagged TER and TER variants in place of wild-type Tetrahymena TER. We found that sequence substitutions in nontemplate regions of TER altered telomere length maintenance in vivo, with an increase or decrease in the set point for telomere length homeostasis. We also characterized the in vitro activity of the telomerase holoenzymes reconstituted with TER variants, following RNA-based RNP affinity purification from cell extracts. We found that nontemplate sequence substitutions imposed specific defects in the fidelity and processivity of template use. These findings demonstrate nontemplate functions of TER that are critical for the telomerase holoenzyme catalytic cycle and for proper telomere length maintenance in vivo.Telomeres protect authentic chromosome termini from aberrant resection or rearrangement. In most eukaryotes, a stable telomere is composed of a tandem array of simple-sequence repeats assembled into specialized chromatin (37). The overall length of the telomeric simple-sequence repeat tract is maintained in a dynamic balance of sequence loss and addition. To compensate for the inevitable loss from incomplete replication by DNA-dependent DNA polymerases, telomeres recruit the telomerase ribonucleoprotein (RNP) reverse transcriptase in coordination with other DNA replication factors (18). Telomerase copies a defined sequence within its integral RNA subunit (TER) to extend chromosome 3Ј ends. Sequence substitutions within the TER template can rapidly impose defects in cell division and viability due to the synthesis of altered repeats (6).During a catalytic cycle of repeat synthesis, the telomerase active site must proceed through a remarkable number of distinct states of template positioning and pairing (Fig. 1A). Substrates unable to base-pair with the template are extended in alignment with the template 3Ј end at its "default" position, defined as the position of template in the absence of a template-primer hybrid (41). For Tetrahymena thermophila TER, default template positioning places C49 in the active site (Fig. 1A, top). As deoxynucleoside triphosphates (dNTPs) are added, a template-product hybrid forms, and successively more 5Ј positions of unpaired template must thread into the active site (Fig. 1A, middle). Product synthesis halts at the template 5Ј boundary, corresponding to position C43 of T. thermophila TER (Fig. 1A, bottom). Upon eventual dissociation of the template-product hybrid, the liberated template repositions to recover its default placement. Product either dissociates from the enzyme or r...

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Identification of an essential proximal sequence element in the promoter of the telomerase RNA gene of Tetrahymena thermophila

Cited by 16 publications

References 38 publications

Combination of Two Regulatory Elements in the Tetrahymena thermophila HSP70-1 Gene Controls Heat Shock Activation

Combination of Two Regulatory Elements in the Tetrahymena thermophila HSP70-1 Gene Controls Heat Shock Activation

Tetrahymena ORC contains a ribosomal RNA fragment that participates in rDNA origin recognition

Biological and Biochemical Functions of RNA in the Tetrahymena Telomerase Holoenzyme

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