Mapping of DNA Binding Sites in the Tetrahymena Telomerase Holoenzyme Proteins by UV Cross-Linking and Mass Spectrometry

Rosenfeld, Karin Kertesz; Ziv, Tamar; Goldin, Svetlana; Glaser, Fabian; Manor, Haim

doi:10.1016/j.jmb.2011.04.040

Cited by 11 publications

(19 citation statements)

References 40 publications

(64 reference statements)

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“…5). Telomerase product DNA can be crosslinked to p45 as well as the TERT TEN domain 31,40 , which suggests that at least some orientations of 7-1-4 must bring p45 close to DNA.…”

Section: -1-4 Subcomplex Has Multiple Orientationsmentioning

confidence: 99%

The architecture of Tetrahymena telomerase holoenzyme

Jiang

Miracco

Hong

et al. 2013

Nature

103

189

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Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the 7 proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labeling. Fitting with high-resolution structures reveals the organization of TERT, TER, and p65 in the RNP catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75-p19-p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function.

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“…5). Telomerase product DNA can be crosslinked to p45 as well as the TERT TEN domain 31,40 , which suggests that at least some orientations of 7-1-4 must bring p45 close to DNA.…”

Section: -1-4 Subcomplex Has Multiple Orientationsmentioning

confidence: 99%

The architecture of Tetrahymena telomerase holoenzyme

Jiang

Miracco

Hong

et al. 2013

Nature

103

189

View full text Add to dashboard Cite

show abstract

“…We suggest as a working model that one or more subunits of Tetrahymena TASC, the human protein TPP1, and the yeast protein Est3 are architecturally and functionally analogous in their respective telomerase holoenzymes: holoenzyme protein interaction(s) with the TEN domain contribute allosteric stabilization of the elongation-competent architecture of the catalytic core RNP and could also extend the direct surface of DNA contact (32,33). Better understanding of the elongating telomerase holoenzyme architecture remains a critical goal for future studies.…”

Section: Discussionmentioning

confidence: 99%

Roles of Telomerase Reverse Transcriptase N-terminal Domain in Assembly and Activity of Tetrahymena Telomerase Holoenzyme

Eckert

Collins

2012

Journal of Biological Chemistry

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Background: Telomerase synthesizes single-stranded telomeric repeats. Results: The TERT N-terminal domain coordinates telomerase holoenzyme subunit association, DNA synthesis initiation, and rate of elongation. Conclusion: TERT N-terminal domain recruitment of holoenzyme proteins increases overall activity and confers repeat addition processivity. Significance: New understanding of DNA handling by a telomerase holoenzyme illuminates mechanisms for processive synthesis of single-stranded telomeric repeats.

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“…Cross-linking can be achieved by several approaches such as photo-induced and chemical cross-linking [46][47][48]. The latter is discussed here in more detail.…”

Section: Cross-linking Msmentioning

confidence: 99%

Mass spectrometric methods to analyze the structural organization of macromolecular complexes

Rajabi

Ashcroft

Radford

2015

Methods

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With the development of soft ionization techniques such as electrospray ionization (ESI), mass spectrometry (MS) has found widespread application in structural biology. The ability to transfer large biomolecular complexes intact into the gas-phase, combined with the low sample consumption and high sensitivity of MS, has made ESI-MS a method of choice for the characterization of macromolecules. This paper describes the application of MS to study large non-covalent complexes. We categorize the available techniques in two groups. First, solution-based techniques in which the biomolecules are labeled in solution and subsequently characterized by MS. Three MS-based techniques are discussed, namely hydroxyl radical footprinting, cross-linking and hydrogen/deuterium exchange (HDX) MS. In the second group, MS-based techniques to probe intact biomolecules in the gas-phase, e.g. side-chain microsolvation, HDX and ion mobility spectrometry are discussed. Together, the approaches place MS as a powerful methodology for an ever growing plethora of structural applications.

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Mapping of DNA Binding Sites in the Tetrahymena Telomerase Holoenzyme Proteins by UV Cross-Linking and Mass Spectrometry

Cited by 11 publications

References 40 publications

The architecture of Tetrahymena telomerase holoenzyme

The architecture of Tetrahymena telomerase holoenzyme

Roles of Telomerase Reverse Transcriptase N-terminal Domain in Assembly and Activity of Tetrahymena Telomerase Holoenzyme

Mass spectrometric methods to analyze the structural organization of macromolecular complexes

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