Studies on RNA-protein interactions and enzyme activity by mutational analysis of telomerase.
The protein component of telomerase (TERT: telomerase reverse transcriptase) contains a domain (RID2: RNA interaction domain 2), which interacts with the RNA component of telomerase (TR: telomerase RNA). RID2 includes a telomerase-specific motif (T-motif), which is highly conserved among species. But, the role of T-motif is not clearly understood. To elucidate the role of T-motif in the telomerase activity and the TERT-TR interaction, we designed two systems including full-length human TERT (hTERT) proteins for telomerase activity and RID2 domain peptides for binding activity. Variants of hTERT, in which a highly conserved amino acid residue in T-motif was replaced with an alanine residue, were expressed in the in vitro translation system of rabbit reticulocyte lysate. We examined telomerase activity of the hTERT variants by the stretch-PCR method. T564A, E565A, and W581A variants did not show remarkably decreased activity suggesting that these amino acid residues are not important for the enzymatic activity, although they are conserved with 89-100% identity. Next, we tried construction of an experimental system for examination of RNA-binding activity of RID2 variants with the same mutations in T-motif. The wild-type RID2 peptide with His-tags was prepared by expression in an E. coli system and purified with an affinity column. The binding activity to human TR (hTR) was examined by the EMSA (electrophoretic mobility shift assay) method. The RID2 (aa 300-617) peptide showed binding to the 3'half molecule of hTR but not to the 5'-half molecule. The RID2 variants are being prepared and analyzed in this system.
Ohsako
Yoshihisa
Y
Department of Environment and Natural Sciences, Graduate School of Environment and information Sciences, Yokohama National University, 79-7 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan.
Tsutsumi
Kota
K
Li
Xianglan
X
Kurihara
Yasuyuki
Y
Uesugi
Seiichi
S
D006801:Humans; D009154:Mutation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism*; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C422952:telomerase RNA; D012313:RNA; C509186:TERT protein, human; D019098:Telomerase