title: Tetrahymena telomerase holoenzyme assembly, activation, and inhibition by domains of the p50 central hub.
abstract: The eukaryotic reverse transcriptase, telomerase, adds tandem telomeric repeats to chromosome ends to promote genome stability. The fully assembled telomerase holoenzyme contains a ribonucleoprotein (RNP) catalytic core and additional proteins that modulate the ability of the RNP catalytic core to elongate telomeres. Electron microscopy (EM) structures of Tetrahymena telomerase holoenzyme revealed a central location of the relatively uncharacterized p50 subunit. Here we have investigated the biochemical and structural basis for p50 function. We have shown that the p50-bound RNP catalytic core has a relatively low rate of tandem repeat synthesis but high processivity of repeat addition, indicative of high stability of enzyme-product interaction. The rate of tandem repeat synthesis is enhanced by p50-dependent recruitment of the holoenzyme single-stranded DNA binding subunit, Teb1. An N-terminal p50 domain is sufficient to stimulate tandem repeat synthesis and bridge the RNP catalytic core, Teb1, and the p75 subunit of the holoenzyme subcomplex p75/p19/p45. In cells, the N-terminal p50 domain assembles a complete holoenzyme that is functional for telomere maintenance, albeit at shortened telomere lengths. Also, in EM structures of holoenzymes, only the N-terminal domain of p50 is visible. Our findings provide new insights about subunit and domain interactions and functions within the Tetrahymena telomerase holoenzyme.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
authors.forename: Kyungah
authors.initials: K
authors.lastname: Hong
authors.forename: Heather
authors.initials: H
authors.lastname: Upton
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Miracco
authors.forename: Jiansen
authors.initials: J
authors.lastname: Jiang
authors.forename: Z Hong
authors.initials: ZH
authors.lastname: Zhou
authors.forename: Juli
authors.initials: J
authors.lastname: Feigon
authors.forename: Kathleen
authors.initials: K
authors.lastname: Collins
mesh_terms: D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D021621:Imaging, Three-Dimensional; D008854:Microscopy, Electron; D008958:Models, Molecular; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D013768:Tetrahymena / Q000201:enzymology*
chemical_list: D020035:Holoenzymes; D021122:Protein Subunits; D015800:Protozoan Proteins; D012261:Ribonucleoproteins
references.citation: Cold Spring Harb Perspect Biol. 2013 May 01;5(5):a010405
references.pmid: 23543032
references.citation: Mol Cell. 2011 May 6;42(3):308-18
references.pmid: 21514196
references.citation: Mol Cell. 2009 Nov 25;36(4):609-19
references.pmid: 19941821
references.citation: J Biol Chem. 2010 May 28;285(22):16434-43
references.pmid: 20363756
references.citation: Proc Natl Acad Sci U S A. 2011 Dec 20;108(51):20357-61
references.pmid: 22143754
references.citation: Annu Rev Genet. 2008;42:301-34
references.pmid: 18680434
references.citation: Mutat Res. 2012 Feb 1;730(1-2):12-9
references.pmid: 21945241
references.citation: Nat Med. 2006 Oct;12(10):1133-8
references.pmid: 17024208
references.citation: Methods Cell Biol. 2012;109:347-55
references.pmid: 22444151
references.citation: Genes Dev. 2004 May 15;18(10):1107-18
references.pmid: 15131081
references.citation: RNA. 2012 Oct;18(10):1747-59
references.pmid: 22875809
references.citation: Nat Rev Genet. 2012 Oct;13(10):693-704
references.pmid: 22965356
references.citation: Methods Cell Biol. 2012;109:327-45
references.pmid: 22444150
references.citation: Mol Cell. 2000 Oct;6(4):827-37
references.pmid: 11090621
references.citation: Nat Rev Mol Cell Biol. 2013 Feb;14(2):69-82
references.pmid: 23299958
references.citation: J Biol Chem. 2001 Feb 16;276(7):4863-71
references.pmid: 11096070
references.citation: Methods Cell Biol. 2012;109:1-7
references.pmid: 22444140
references.citation: Cold Spring Harb Perspect Biol. 2011 May 01;3(5):
references.pmid: 20660025
references.citation: Semin Cancer Biol. 2011 Dec;21(6):349-53
references.pmid: 22015685
references.citation: Mutat Res. 2012 Feb 1;730(1-2):3-11
references.pmid: 22093366
references.citation: J Microsc. 1987 May;146(Pt 2):113-36
references.pmid: 3302267
references.citation: Cell. 2012 Jul 6;150(1):39-52
references.pmid: 22748632
references.citation: J Cell Mol Med. 2011 Jan;15(1):3-13
references.pmid: 21122064
references.citation: Curr Opin Chem Biol. 2011 Oct;15(5):643-8
references.pmid: 21816660
references.citation: Nature. 2013 Apr 11;496(7444):187-92
references.pmid: 23552895
references.citation: Nat Rev Mol Cell Biol. 2010 Mar;11(3):171-81
references.pmid: 20125188
references.citation: Mol Cell Biol. 2007 Mar;27(6):2074-83
references.pmid: 17220281
references.citation: Trends Biochem Sci. 2012 Sep;37(9):391-9
references.pmid: 22727244
references.citation: Curr Opin Chem Biol. 2011 Dec;15(6):845-52
references.pmid: 22057212