scholarly journals Essential Regions of Saccharomyces cerevisiae Telomerase RNA: Separate Elements for Est1p and Est2p Interaction

2002 ◽  
Vol 22 (7) ◽  
pp. 2366-2374 ◽  
Author(s):  
April J. Livengood ◽  
Arthur J. Zaug ◽  
Thomas R. Cech
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Dna Synthesis ◽  
Active Site ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Protein Subunits ◽  
Essential Region ◽  
Selection For

ABSTRACT The Saccharomyces cerevisiae telomerase RNA subunit is encoded by the TLC1 gene. A selection for viable alleles of TLC1 RNA from a large library of random deletion alleles revealed that less than half (∼0.5 kb of the ∼1.3-kb RNA) is required for telomerase function in vivo. The main essential region (430 nucleotides), which contains the template for telomeric DNA synthesis, was required for coimmunoprecipitation with Est1p and Est2p. Furthermore, the subregion required for interaction with Est1p, the telomerase recruitment subunit, differed from those required for interaction with Est2p, the reverse transcriptase subunit. Two regions of the RNA distant from the template in the nucleotide sequence were required for Est2p binding, but the template itself was not. Having the RNA secured to the protein away from the template is proposed to facilitate the translocation of the RNA template through the active site. More generally, our results support a role for the telomerase RNA serving as a scaffold for binding key protein subunits.

scholarly journals Repositioning the Sm-Binding Site in Saccharomyces cerevisiae Telomerase RNA Reveals RNP Organizational Flexibility and Sm-Directed 3′-End Formation

2020 ◽  
Vol 6 (1) ◽  
pp. 9 ◽  
Author(s):  
Evan P. Hass ◽  
David C. Zappulla
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Protein Subunits ◽  
Yeast Telomerase ◽  
Rna Biogenesis ◽  
Native Site ◽  
Processing Mechanism

Telomerase RNA contains a template for synthesizing telomeric DNA and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In Saccharomyces cerevisiae, the telomerase RNA, TLC1, is bound by the Sm7 protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)–) TLC1 isoform. However, it remains unclear (1) whether Sm7 retains this function when its binding site is repositioned within TLC1, as has been shown for other TLC1-binding telomerase subunits, and (2) how Sm7 stabilizes poly(A)– TLC1. Here, we first show that Sm7 can stabilize poly(A)– TLC1 even when its binding site is repositioned via circular permutation to several different positions within TLC1, further supporting the conclusion that the telomerase holoenzyme is organizationally flexible. Next, we show that when an Sm site is inserted 5′ of its native position and the native site is mutated, Sm7 stabilizes shorter forms of poly(A)– TLC1 in a manner corresponding to how far upstream the new site was inserted, providing strong evidence that Sm7 binding to TLC1 controls where the mature poly(A)– 3′ is formed by directing a 3′-to-5′ processing mechanism. In summary, our results show that Sm7 and the 3′ end of yeast telomerase RNA comprise an organizationally flexible module within the telomerase RNP and provide insights into the mechanistic role of Sm7 in telomerase RNA biogenesis.

scholarly journals Repositioning the Sm-binding site in S. cerevisiae telomerase RNA reveals RNP organizational flexibility and Sm-directed 3′-end formation

2017 ◽  
Author(s):  
Evan P. Hass ◽  
David C. Zappulla
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Strong Evidence ◽  
Binding Sites ◽  
Telomerase Rna ◽  
Telomeric Dna ◽  
Protein Subunits ◽  
Yeast Telomerase ◽  
Rna Biogenesis

ABSTRACTTelomerase RNA contains a template for synthesizing telomeric DNA by reverse transcription and has been proposed to act as a flexible scaffold for holoenzyme protein subunits in the RNP. In Saccharomyces cerevisiae, the telomerase subunits Est1 and Ku bind to the telomerase RNA, TLC1, and it has been shown that these proteins still function when their binding sites are repositioned within the RNA. TLC1 is also bound by the Sm7 protein complex, which is required for stabilization of the predominant, non-polyadenylated (poly(A)–) TLC1 isoform. Here, we first show that Sm7 can perform this function even when its binding site is repositioned via circular permutation to several different positions within TLC1, further supporting the conclusion that the telomerase holoenzyme is organizationally flexible. Next, we tested the hypothesis that the location of the Sm7-binding site relative to the 3′ end is contrastingly important. When we moved the Sm site to locations 5′ of its native position, we observed that this stabilized shorter forms of poly(A)– TLC1 in a manner precisely corresponding to how far upstream the Sm site was moved. This provides strong evidence that the location of Sm7 binding to TLC1 controls where the mature poly(A)– 3′ end is formed. In summary, our results show that Sm7 and the 3′ end of yeast telomerase RNA comprise an organizationally flexible module within the telomerase RNP and provide insights into the mechanistic role of Sm7 in telomerase RNA biogenesis.

scholarly journals Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase.

1996 ◽  
Vol 16 (1) ◽  
pp. 66-75 ◽  
Author(s):  
D Gilley ◽  
E H Blackburn
Keyword(s):  
Active Site ◽  
Tetrahymena Thermophila ◽  
Telomerase Rna ◽  
Specific Interactions ◽  
Wild Type ◽  
Specific Primer ◽  
Telomeric Dna ◽  
Template Sequence ◽  
New Evidence

The ribonucleoprotein enzyme telomerase is a specialized reverse transcriptase that synthesizes telomeric DNA by copying a template sequence within the telomerase RNA. Here we analyze the actions of telomerase from Tetrahymena thermophila assembled in vivo with mutated or wild-type telomerase RNA to define further the roles of particular telomerase RNA residues involved in essential enzymatic functions: templating, substrate alignment, and promotion of polymerization. Position 49 of the telomerase RNA defined the 3' templating residue boundary, demonstrating that seven positions, residues 43 to 49, are capable of acting as templating residues. We demonstrate directly that positioning of the primer substrate involves Watson-Crick base pairing between the primer with telomerase RNA residues. Unexpectedly, formation of a Watson-Crick base pair specifically between the primer DNA and telomerase RNA residue 50 is critical in promoting primer elongation. In contrast, mutant telomerase with the cytosine at position 49 mutated to a G exhibited efficient 3' mispair extension. This work provides new evidence for specific primer-telomerase interactions, as well as base-specific interactions involving the telomerase RNA, playing roles in essential active-site functions of telomerase.

scholarly journals Saccharomyces cerevisiae Grx6 and Grx7 Are Monothiol Glutaredoxins Associated with the Early Secretory Pathway

2008 ◽  
Vol 7 (8) ◽  
pp. 1415-1426 ◽  
Author(s):  
Alicia Izquierdo ◽  
Celia Casas ◽  
Ulrich Mühlenhoff ◽  
Christopher Horst Lillig ◽  
Enrique Herrero
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Inhibitory Effect ◽  
Protein Accumulation ◽  
Oxidoreductase Activity ◽  
Early Secretory Pathway ◽  
Glycosylation Inhibitor

ABSTRACT Saccharomyces cerevisiae Grx6 and Grx7 are two monothiol glutaredoxins whose active-site sequences (CSYS and CPYS, respectively) are reminiscent of the CPYC active-site sequence of classical dithiol glutaredoxins. Both proteins contain an N-terminal transmembrane domain which is responsible for their association to membranes of the early secretory pathway vesicles, facing the luminal side. Thus, Grx6 localizes at the endoplasmic reticulum and Golgi compartments, while Grx7 is mostly at the Golgi. Expression of GRX6 is modestly upregulated by several stresses (calcium, sodium, and peroxides) in a manner dependent on the Crz1-calcineurin pathway. Some of these stresses also upregulate GRX7 expression under the control of the Msn2/4 transcription factor. The N glycosylation inhibitor tunicamycin induces the expression of both genes along with protein accumulation. Mutants lacking both glutaredoxins display reduced sensitivity to tunicamycin, although the drug is still able to manifest its inhibitory effect on a reporter glycoprotein. Grx6 and Grx7 have measurable oxidoreductase activity in vivo, which is increased in the presence of tunicamycin. Both glutaredoxins could be responsible for the regulation of the sulfhydryl oxidative state at the oxidant conditions of the early secretory pathway vesicles. However, the differences in location and expression responses against stresses suggest that their functions are not totally overlapping.

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 Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (8) ◽  
pp. 1440-1448 ◽  
Author(s):  
M Johnston ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Base Pair ◽  
Transcription Initiation ◽  
Base Pairs ◽  
Regulation Of Expression ◽  
Notable Feature ◽  
His3 Gene

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.

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.

Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae

1984 ◽  
Vol 4 (8) ◽  
pp. 1440-1448
Author(s):  
M Johnston ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Base Pair ◽  
Transcription Initiation ◽  
Base Pairs ◽  
Regulation Of Expression ◽  
Notable Feature ◽  
His3 Gene

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.

scholarly journals FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (10) ◽  
pp. 5968-5975 ◽  
Author(s):  
C M Alarcón ◽  
J Heitman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Conformational Changes ◽  
Active Site ◽  
Feedback Inhibition ◽  
Immunosuppressive Drugs ◽  
Intermediate Step ◽  
Active Site Residues ◽  
Peptidyl Prolyl Isomerase

The peptidyl-prolyl isomerase FKBP12 was originally identified as the intracellular receptor for the immunosuppressive drugs FK506 (tacrolimus) and rapamycin (sirolimus). Although peptidyl-prolyl isomerases have been implicated in catalyzing protein folding, the cellular functions of FKBP12 in Saccharomyces cerevisiae and other organisms are largely unknown. Using the yeast two-hybrid system, we identified aspartokinase, an enzyme that catalyzes an intermediate step in threonine and methionine biosynthesis, as an in vivo binding target of FKBP12. Aspartokinase also binds FKBP12 in vitro, and drugs that bind the FKBP12 active site, or mutations in FKBP12 surface and active site residues, disrupt the FKBP12-aspartokinase complex in vivo and in vitro.fpr1 mutants lacking FKBP12 are viable, are not threonine or methionine auxotrophs, and express wild-type levels of aspartokinase protein and activity; thus, FKBP12 is not essential for aspartokinase activity. The activity of aspartokinase is regulated by feedback inhibition by product, and genetic analyses reveal that FKBP12 is important for this feedback inhibition, possibly by catalyzing aspartokinase conformational changes in response to product binding.

scholarly journals Yeast Telomerase RNA Flexibly Scaffolds Protein Subunits: Results and Repercussions

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2750 ◽  
Author(s):  
David C. Zappulla
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Noncoding Rna ◽  
Noncoding Rnas ◽  
Telomerase Rna ◽  
Protein Subunits ◽  
Rna Molecules ◽  
Yeast Telomerase ◽  
Opportune Time ◽  
Rnp Complex ◽  
Functional Features

It is said that “hindsight is 20-20,” so, given the current year, it is an opportune time to review and learn from experiences studying long noncoding RNAs. Investigation of the Saccharomyces cerevisiae telomerase RNA, TLC1, has unveiled striking flexibility in terms of both structural and functional features. Results support the “flexible scaffold” hypothesis for this 1157-nt telomerase RNA. This model describes TLC1 acting as a tether for holoenzyme protein subunits, and it also may apply to a plethora of RNAs beyond telomerase, such as types of lncRNAs. In this short perspective review, I summarize findings from studying the large yeast telomerase ribonucleoprotein (RNP) complex in the hope that this hindsight will sharpen foresight as so many of us seek to mechanistically understand noncoding RNA molecules from vast transcriptomes.

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