Requirement for C-terminal extension to the RNA binding domain for efficient RNA binding by ribosomal protein L2.
Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.
Hayashi
Takeshi
T
Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Tahara
Maino
M
Iwasaki
Kenta
K
Kouzuma
Yoshiaki
Y
Kimura
Makoto
M
D001411:Geobacillus stearothermophilus / Q000378:metabolism; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C029466:ribosomal protein L2