scholarly journals The yeast telomerase RNA, TLC1, participates in two distinct modes of TLC1-TLC1 association processes in vivo

2015 ◽  
Author(s):  
Tet Matsuguchi ◽  
Elizabeth Blackburn
Keyword(s):  
Nuclear Periphery ◽  
Repeat Sequence ◽  
Telomeric Repeat ◽  
G1 Phase ◽  
Telomerase Rna ◽  
Reverse Transcriptase Domain ◽  
Core Enzyme ◽  
Yeast Telomerase ◽  
Enzymatic Activation

Telomerase core enzyme minimally consists of the telomerase reverse transcriptase domain-containing protein (Est2 in budding yeast S. cerevisiae) and telomerase RNA, which contains the template specifying the telomeric repeat sequence synthesized. Here we report that in vivo, a fraction of S. cerevisiae telomerase RNA (TLC1) molecules form complexes containing at least two molecules of TLC1, via two separable modes: one requiring a sequence in the 3’ region of the immature TLC1 precursor and the other requiring Ku and Sir4. Such physical TLC1-TLC1 association peaked in G1 phase and did not require telomere silencing, telomere tethering to the nuclear periphery, telomerase holoenzyme assembly, or detectable Est2-Est2 protein association. These data indicate that TLC1-TLC1 associations reflect processes occurring during telomerase biogenesis; we propose that TLC1-TLC1 associations and subsequent reorganization may be regulatory steps in telomerase enzymatic activation.

scholarly journals The yeast telomerase RNA, TLC1, participates in two distinct modes of TLC1-TLC1 association processesin vivo

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1534
Author(s):  
Tet Matsuguchi ◽  
Elizabeth Blackburn
Keyword(s):  
Nuclear Periphery ◽  
Repeat Sequence ◽  
Telomeric Repeat ◽  
G1 Phase ◽  
Telomerase Rna ◽  
Reverse Transcriptase Domain ◽  
Core Enzyme ◽  
Yeast Telomerase ◽  
Enzymatic Activation

Telomerase core enzyme minimally consists of the telomerase reverse transcriptase domain-containing protein (Est2 in budding yeastS. cerevisiae) and telomerase RNA, which contains the template specifying the telomeric repeat sequence synthesized. Here we report thatin vivo, a fraction ofS. cerevisiaetelomerase RNA (TLC1) molecules form complexes containing at least two molecules of TLC1, via two separable modes: one requiring a sequence in the 3′ region of the immature TLC1 precursor and the other requiring Ku and Sir4. Such physical TLC1-TLC1 association peaked in G1 phase and did not require telomere silencing, telomere tethering to the nuclear periphery, telomerase holoenzyme assembly, or detectable Est2-Est2 protein association. These data indicate that TLC1-TLC1 associations reflect processes occurring during telomerase biogenesis; we propose that TLC1-TLC1 associations and subsequent reorganization may be regulatory steps in telomerase enzymatic activation.

scholarly journals The yeast telomerase RNA, TLC1, participates in two distinct modes of TLC1-TLC1 association processes in vivo

2015 ◽  
Author(s):  
Tet Matsuguchi ◽  
Elizabeth Blackburn
Keyword(s):  
Nuclear Periphery ◽  
Repeat Sequence ◽  
Telomeric Repeat ◽  
G1 Phase ◽  
Telomerase Rna ◽  
Reverse Transcriptase Domain ◽  
Core Enzyme ◽  
Yeast Telomerase ◽  
Enzymatic Activation

Telomerase core enzyme minimally consists of the telomerase reverse transcriptase domain-containing protein (Est2 in budding yeast S. cerevisiae) and telomerase RNA, which contains the template specifying the telomeric repeat sequence synthesized. Here we report that in vivo, a fraction of S. cerevisiae telomerase RNA (TLC1) molecules form complexes containing at least two molecules of TLC1, via two separable modes: one requiring a sequence in the 3’ region of the immature TLC1 precursor and the other requiring Ku and Sir4. Such physical TLC1-TLC1 association peaked in G1 phase and did not require telomere silencing, telomere tethering to the nuclear periphery, telomerase holoenzyme assembly, or detectable Est2-Est2 protein association. These data indicate that TLC1-TLC1 associations reflect processes occurring during telomerase biogenesis; we propose that TLC1-TLC1 associations and subsequent reorganization may be regulatory steps in telomerase enzymatic activation.

A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro

2005 ◽  
Vol 12 (12) ◽  
pp. 1072-1077 ◽  
Author(s):  
David C Zappulla ◽  
Karen Goodrich ◽  
Thomas R Cech
Keyword(s):  
Telomerase Rna ◽  
Yeast Telomerase

scholarly journals A 4-base pair core-enclosing helix in telomerase RNA is essential and binds to the TERT catalytic protein subunit

2020 ◽  
Author(s):  
Melissa A. Mefford ◽  
Evan P. Hass ◽  
David C. Zappulla
Keyword(s):  
Telomerase Activity ◽  
Genome Replication ◽  
Telomerase Rna ◽  
Protein Subunit ◽  
Base Pairs ◽  
Binding Interaction ◽  
Catalytic Core ◽  
Yeast Telomerase

ABSTRACTThe telomerase RNP counters the chromosome end-replication problem, completing genome replication to prevent cellular senescence in yeast, humans, and most other eukaryotes. The telomerase RNP core enzyme is composed of a dedicated RNA subunit and a reverse transcriptase (TERT). Although the majority of the 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, is rapidly evolving, the central catalytic core is largely conserved, containing the template, template-boundary helix, pseudoknot, and core-enclosing helix (CEH). Here, we show that 4-base pairs of core-enclosing helix is required for telomerase to be active in vitro and to maintain yeast telomeres in vivo, whereas ΔCEH, 1-bp, and 2-bp alleles do not support telomerase function. Using the CRISPR/dCas9-based “CARRY two-hybrid” assay to assess binding of our CEH mutant RNAs to TERT, we find that the 4-bp CEH RNA binds to TERT, but the shorter-CEH constructs do not, consistent with the telomerase activity and in vivo complementation results. Thus, the CEH is essential in yeast telomerase RNA because it is needed to bind TERT to form the core RNP enzyme. Although the 8 nucleotides that form this 4-bp stem at the base of the CEH are nearly invariant among Saccharomyces species, our results with sequence-randomized and truncated-CEH helices strongly suggest that this binding interaction with TERT is dictated more by secondary than primary structure. In summary, we have mapped an essential binding site in telomerase RNA for TERT that is crucial to form the catalytic core of this biomedically important RNP enzyme.

scholarly journals Stiffened yeast telomerase RNA supports RNP function in vitro and in vivo

RNA ◽  
2012 ◽  
Vol 18 (9) ◽  
pp. 1666-1678 ◽  
Author(s):  
K. J. Lebo ◽  
D. C. Zappulla
Keyword(s):  
Telomerase Rna ◽  
Yeast Telomerase

scholarly journals Telomere Formation by Rap1p Binding Site Arrays Reveals End-Specific Length Regulation Requirements and Active Telomeric Recombination

2001 ◽  
Vol 21 (23) ◽  
pp. 8117-8128 ◽  
Author(s):  
Simona Grossi ◽  
Alessandro Bianchi ◽  
Pascal Damay ◽  
David Shore
Keyword(s):  
Binding Sites ◽  
De Novo ◽  
Repeat Sequence ◽  
Independent Manner ◽  
Telomere Repeat ◽  
Length Regulation ◽  
End Capping ◽  
Original Array

ABSTRACT Rap1p, the major telomere repeat binding protein in yeast, has been implicated in both de novo telomere formation and telomere length regulation. To characterize the role of Rap1p in these processes in more detail, we studied the generation of telomeres in vivo from linear DNA substrates containing defined arrays of Rap1p binding sites. Consistent with previous work, our results indicate that synthetic Rap1p binding sites within the internal half of a telomeric array are recognized as an integral part of the telomere complex in an orientation-independent manner that is largely insensitive to the precise spacing between adjacent sites. By extending the lengths of these constructs, we found that several different Rap1p site arrays could never be found at the very distal end of a telomere, even when correctly oriented. Instead, these synthetic arrays were always followed by a short (≈100-bp) “cap” of genuine TG repeat sequence, indicating a remarkably strict sequence requirement for an end-specific function(s) of the telomere. Despite this fact, even misoriented Rap1p site arrays promote telomere formation when they are placed at the distal end of a telomere-healing substrate, provided that at least a single correctly oriented site is present within the array. Surprisingly, these heterogeneous arrays of Rap1p binding sites generate telomeres through a RAD52-dependent fusion resolution reaction that results in an inversion of the original array. Our results provide new insights into the nature of telomere end capping and reveal one way by which recombination can resolve a defect in this process.

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