TY - JOUR
T1 - On the interaction of ribosomal protein L5 with 5S rRNA
AU - Iwasaki, Kenta
AU - Kikukawa, Shingo
AU - Kawamura, Shunsuke
AU - Kouzuma, Yoshiaki
AU - Tanaka, Isao
AU - Kimura, Makoto
N1 - Funding Information:
This work was supported in part by a Grant-in-Aid for Scientiˆc Research from The Ministry of Education, Science, Sports, and Culture of Japan, and by a Grant of Rice Genome Project PR-3003, MAFF, Japan.
PY - 2002
Y1 - 2002
N2 - Ribosomal protein L5, a 5S rRNA binding protein in the large subunit, is composed of a five-stranded antiparallel β-sheet and four α-helices, and folds in a way that is topologically similar to the ribonucleprotein (RNP) domain [Nakashima et al., RNA 7, 692–701, 2001]. The crystal structure of ribosomal protein L5 (BstL5) from Bacillus stearothermophilus suggests that a concave surface formed by an anti-parallel β-sheet and long loop structures are strongly involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurred at β-strands and loop structures in BstL5. The mutation of Lys33 at the β1-strand caused a significant reduction in 5S rRNA binding. In addition, the Arg92, Phe122, and Glu134 mutations on the β2-strand, the α3-β4 loop, and the β4-β5 loop, respectively, resulted in a moderate decrease in the 5S rRNA binding affinity. In contrast, mutation of the conserved residue Pro65 at the β2-strand had little effect on the 5S rRNA binding activity. These results, taken together with previous results, identified Lys33, Asn37, Gln63, and Thr90 on the β-sheet structure, and Phe77 at the β2-β3 loop as critical residues for the 5S rRNA binding. The contribution of these amino acids to 5S rRNA binding was further quantitatively evaluated by surface plasmon resonance (SPR) analysis by the use of BIAcore. The results showed that the amino acids on the β-sheet structure are required to decrease the dissociation rate constant for the BstL5-5S rRNA complex, while those on the loops are to increase the association rate constant for the BstL5-5S rRNA interaction.
AB - Ribosomal protein L5, a 5S rRNA binding protein in the large subunit, is composed of a five-stranded antiparallel β-sheet and four α-helices, and folds in a way that is topologically similar to the ribonucleprotein (RNP) domain [Nakashima et al., RNA 7, 692–701, 2001]. The crystal structure of ribosomal protein L5 (BstL5) from Bacillus stearothermophilus suggests that a concave surface formed by an anti-parallel β-sheet and long loop structures are strongly involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurred at β-strands and loop structures in BstL5. The mutation of Lys33 at the β1-strand caused a significant reduction in 5S rRNA binding. In addition, the Arg92, Phe122, and Glu134 mutations on the β2-strand, the α3-β4 loop, and the β4-β5 loop, respectively, resulted in a moderate decrease in the 5S rRNA binding affinity. In contrast, mutation of the conserved residue Pro65 at the β2-strand had little effect on the 5S rRNA binding activity. These results, taken together with previous results, identified Lys33, Asn37, Gln63, and Thr90 on the β-sheet structure, and Phe77 at the β2-β3 loop as critical residues for the 5S rRNA binding. The contribution of these amino acids to 5S rRNA binding was further quantitatively evaluated by surface plasmon resonance (SPR) analysis by the use of BIAcore. The results showed that the amino acids on the β-sheet structure are required to decrease the dissociation rate constant for the BstL5-5S rRNA complex, while those on the loops are to increase the association rate constant for the BstL5-5S rRNA interaction.
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U2 - 10.1271/bbb.66.103
DO - 10.1271/bbb.66.103
M3 - Article
C2 - 11866091
AN - SCOPUS:0036359054
VL - 66
SP - 103
EP - 109
JO - Bioscience, Biotechnology and Biochemistry
JF - Bioscience, Biotechnology and Biochemistry
SN - 0916-8451
IS - 1
ER -