Minimum length requirement of the alignment domain of human telomerase RNA to sustain catalytic activity in vitro

被引:33
作者
Gavory, G [1 ]
Farrow, M [1 ]
Balasubramanian, S [1 ]
机构
[1] Univ Cambridge, Chem Lab, Cambridge CB2 1EW, England
基金
英国生物技术与生命科学研究理事会;
关键词
D O I
10.1093/nar/gkf575
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Telomeres are essential for genomic stability and cell viability. Telomerase, the enzyme responsible for telomere maintenance, is composed of a reverse transcriptase protein subunit and an integral RNA component which contains the templating domain. In human telomerase, the template region consists of 11 nt (3'-rCAAUCCCAAUC-5') and comprises an alignment domain (italicised) plus a template sequence encoding the telomeric repeat d(GGT TAG). In this study, the alignment domain of human telomerase was systematically reduced from the 3' end and the resultant recombinant enzyme activity was evaluated in vitro. Deletion or substitution of one or two residues from the 3' end of the alignment domain caused only a slight reduction in overall catalytic activity and did not alter the processivity of the enzyme. Deletion or substitution of three or more residues from the 3' end of the alignment domain resulted in total loss of catalytic activity. These results suggest that the two most 3' terminal RNA residues are relevant but not essential for overall activity and that the minimal length requirement of the alignment domain is 3 nt. Furthermore, base pairing between the 3' end of the primer substrate and the first two residues of the alignment domain is also not an absolute requirement for processive synthesis.
引用
收藏
页码:4470 / 4480
页数:11
相关论文
共 55 条
[1]   Analysis of the structure of human telomerase RNA in vivo [J].
Antal, M ;
Boros, É ;
Solymosy, F ;
Kiss, T .
NUCLEIC ACIDS RESEARCH, 2002, 30 (04) :912-920
[2]   Tetrahymena telomerase ribonucleoprotein RNA-protein interactions [J].
Autexier, C ;
Triki, I .
NUCLEIC ACIDS RESEARCH, 1999, 27 (10) :2227-2234
[3]   Mutational analysis of the Tetrahymena telomerase RNA:: identification of residues affecting telomerase activity in vitro [J].
Autexier, C ;
Greider, CW .
NUCLEIC ACIDS RESEARCH, 1998, 26 (03) :787-795
[4]   BOUNDARY ELEMENTS OF THE TETRAHYMENA TELOMERASE RNA TEMPLATE AND ALIGNMENT DOMAINS [J].
AUTEXIER, C ;
GREIDER, CW .
GENES & DEVELOPMENT, 1995, 9 (18) :2227-2239
[5]   FUNCTIONAL RECONSTITUTION OF WILD-TYPE AND MUTANT TETRAHYMENA TELOMERASE [J].
AUTEXIER, C ;
GREIDER, CW .
GENES & DEVELOPMENT, 1994, 8 (05) :563-575
[6]   Human telomerase RNA-protein interactions [J].
Bachand, F ;
Triki, F ;
Autexier, C .
NUCLEIC ACIDS RESEARCH, 2001, 29 (16) :3385-3393
[7]   Polymerization defects within human telomerase are distinct from telomerase RNA and TEP1 binding [J].
Beattie, TL ;
Zhou, W ;
Robinson, MO ;
Harrington, L .
MOLECULAR BIOLOGY OF THE CELL, 2000, 11 (10) :3329-3340
[8]   Reconstitution of human telomerase activity in vitro [J].
Beattie, TL ;
Zhou, W ;
Robinson, MO ;
Harrington, L .
CURRENT BIOLOGY, 1998, 8 (03) :177-180
[9]   Developmentally regulated initiation of DNA synthesis by telomerase: Evidence for factor-assisted de novo telomere formation [J].
Bednenko, J ;
Melek, M ;
Greene, EC ;
Shippen, DE .
EMBO JOURNAL, 1997, 16 (09) :2507-2518
[10]   STRUCTURE AND FUNCTION OF TELOMERES [J].
BLACKBURN, EH .
NATURE, 1991, 350 (6319) :569-573