2003 Fiscal Year Final Research Report Summary
Functional analysis of tRNA splicing endonuclease from Arabidopsis thaliana
| Project/Area Number |
13680766
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | Shimane University |
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Principal Investigator |
AKAMA Kazuhito Shimane University, Faculty of Life and Environmental Science, Research Associate, 生物資源科学部, 助手 (50252896)
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| Project Period (FY) |
2001 – 2003
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| Keywords | tRNA splicing / tRNA splicing endonuclease / intoron / Arabidopsis / transgenic plant / amber suppressor tRNA / Agrobacterium / in vitro transcription / translation |
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| Research Abstract |
tRNA splicing endonuclease plays an important role in the maturation of some tRNAs (tRNA^<Tyr> and tRNA^<Met> in plants) that are interrupted by introns. We have previously isolated two putative genes coding for catalytic subunits of tRNA endonucleases from Arabidopsis (AtSen1 and AtSen2). To determine the properties of the products of these genes, transgenic Arabidopsis plants expressing them were produced : AtSen1 or AtSen2 cDNA or one of these cDNAs with substitution of a His codon encoding one amino acid of the catalytic triad by an Ala codon in position 156 in each ORF (designated AtSen1H156A and AtSen2H156A) were fused to the CaMV 35S promoter in order to overexpress them in plant cells. Plant lines overexpressing each gene were produced using Agrobacterium. Northern analysis of the total tRNAs isolated from these four different plant lines revealed that a significant level of intron-containing pre-tRNA^<Met> was accumulated only in AtSen1H156A plants. This finding suggests that
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AtSen1 by itself (but not AtSen2) has the ability to cleave the tRNA at the 5' and 3' splice sites. In order to confirm this conclusion, we compared the efficiency of the translational suppression of an amber codon in gusA mRNA via suppressor tRNA^<Met> transiently expressed in callus tissues from these transgenic plants and wild-type plants. As expected, relatively lower GUS activity was observed only in AtSen1H156A plants, probably because of a decrease in the translational suppression of the amber codon in the co-expressed gusA mRNA.
In parallel, protein synthesis (AtSen1 and AtSen2) was performed in vitro using the RTS kit (Roche). These proteins were purified with the Ni-NTA resin. In vitro splicing assay of ^<32?P labeled template (pNtY9*T7M1 that carries bulge-helix-bulge motif (8H8)) was done in the presence of AtSen1 or AtSen2. This result indicated that each catalytic subunit, AtSen1 or AtSen2, can independently cleave both 5' and 3' splice sites in the BHB structure of artificial plant pre-tRNA^<Tyr>. However, a typical intron-containing pre-tRNA (i.e., loop-helix-bulge motif) seen in plants was poor substrates in the presence of one of the subunits or both of them, which may imply requirement of additional subunits homologous to yeast Sen54p and/or Sen15p even in plant pre-tRNA splicing. Isolation of these putative subunits is now in progress. Less
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