| Research Abstract |
The GTPase-associated domain in 23/28S rRNA is one of the most highly conserved functional regions throughout all organisms. We detected a unique nucleotide sequence within the GTPase-associated domain in silkworm species, in which the bases at positions 1094 and 1098 (numbering from Escherichia coli 23S rRNA) are C and G instead of the otherwise universally conserved bases U and A, respectively. These changes were also observed in four other moths, but not in organisms other than the moths. The RNA fragment covering the silkworm GTPase-associated domain showed two smearing bands in native gel electrophoresis in the absence of ribosomal proteins. However, these bands shifted to a single clear band, wheh silkworm ribosomal proteins P0, P1, P2, and eL12 bound to the RNA, suggesting that the GTPase-associated domain of silkworm 28S rRNA has a labile structural feature that is stabilized by binding of the ribosomal proteins. In order to know the functional significance of the covariant bas
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e change in the silkworm rRNA, we attempted the introduction of C-1094 and G-1098 into E. coli ribosomes by using a plasmid carrying E. coli rDNA (rrnB). So far, we detected the mutant rRNA in isolated ribosomes, suggesting that the mutant ribosomes were assembled in vivo. Further analyses of function of the mutant ribosomes were remained.
We also investigated interactions among ribosomal proteins P0, P1, and P2 by native gel electrophoresis. Mixing of P1 and P2 formed the P1-P2 heterodimer. This heterodimer, but neither P1 nor P2 alone, tightly bound to P0 and formed a pentameric complex P0(P1-P2)_2, We confirmed the functions of the ribosomal proteins by incorporating the proteins into E. coli ribosomes, i.e., by replacing E. coli proteins L10-L7/L12 complex and L11 on the GTPase-associated RNA domain with silkworm P0(P1-P2)_2 complex and eL12. The protein replacement caused ribosomal activity with silkworm translation factors. This protein-dependent induction of the ribosomal activity did not occur in the absence either of P1 or P2. These results suggest that formation of P1-P2 heterodimer is crucial for assembly of the proteins on the rRNA domain and for the stabilization of the functional rRNA structure. Less
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