A role for aromatic amino acids in the binding of Xenopus ribosomal protein L5 to 5S rRNA

被引:9
作者
DiNitto, JP [1 ]
Huber, PW [1 ]
机构
[1] Univ Notre Dame, Dept Chem & Biochem, Notre Dame, IN 46556 USA
关键词
D O I
10.1021/bi011439m
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The formation of the Xenopus L5-5S rRNA complex depends on nonelectrostatic interactions. Fluorescence assays with 1-anilino-8-naphthalenesulfonate demonstrate that a hydrophobic region on L5 becomes exposed upon removal of bound 5S rRNA by treatment with ribonucleases. Several conserved aromatic amino acids, mostly tyrosines, were identified by comparative sequence analysis and changed individually to alanine. Substitution with alanine at any of three positions, Y86, Y99, or Y226, essentially abolishes RNA-binding activity, whereas those made at Y95 and Y207 have more modest effects. Replacement with phenylalanine at Y86 and Y226 does not change binding affinity, indicating that the aromatic ring of the side chain, not the hydroxyl group, is the critical functionality for binding. Alternatively, the phenolic hydroxyls at Y99 and Y207 do contribute to binding. The structural integrity of the mutant proteins was assessed using thermal denaturation and limited digestion with proteases. The T-m of Y99A is 10 degreesC lower than that of the wild-type protein, and there are some differences in the protease digestion patterns that together indicate the structure of this mutant has been significantly perturbed. The structures of the other variants are not detectably different from the wild-type protein. These results provide evidence that intermolecular stacking interactions involving at least two tyrosine residues, Y86 and Y226, are necessary for formation of the L5-5S rRNA complex and can account, at least in part, for the contribution nonelectrostatic interactions make to the free energy of binding.
引用
收藏
页码:12645 / 12653
页数:9
相关论文
共 43 条
  • [21] NMR structure of the bacteriophage λ N peptide/boxB RNA complex:: Recognition of a GNRA fold by an arginine-rich motif
    Legault, P
    Li, J
    Mogridge, J
    Kay, LE
    Greenblatt, J
    [J]. CELL, 1998, 93 (02) : 289 - 299
  • [22] The participation of 5S rRNA in the co-translational formation of a eukaryotic 5S ribonucleoprotein complex
    Lin, EL
    Lin, SW
    Lin, A
    [J]. NUCLEIC ACIDS RESEARCH, 2001, 29 (12) : 2510 - 2516
  • [23] Mao HY, 1999, NAT STRUCT BIOL, V6, P1139
  • [24] Distinct domains in ribosomal protein L5 mediate 5 S rRNA binding and nucleolar localization
    Michael, WM
    Dreyfuss, G
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (19) : 11571 - 11574
  • [25] RNA-PROTEIN INTERACTIONS - DIVERSE MODES OF RECOGNITION
    MORAS, D
    POTERSZMAN, A
    [J]. CURRENT BIOLOGY, 1995, 5 (03) : 249 - 251
  • [26] A role for ribosomal protein L5 in the nuclear import of 5S rRNA in Xenopus oocytes
    Murdoch, KJ
    Allison, LA
    [J]. EXPERIMENTAL CELL RESEARCH, 1996, 227 (02) : 332 - 343
  • [27] Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding
    Nakashima, T
    Yao, M
    Kawamura, S
    Iwasaki, K
    Kimura, M
    Tanaka, I
    [J]. RNA, 2001, 7 (05) : 692 - 701
  • [28] Novokhatny V, 1997, PROTEIN SCI, V6, P141
  • [29] CRYSTAL-STRUCTURE AT 1.92 ANGSTROM RESOLUTION OF THE RNA-BINDING DOMAIN OF THE U1A SPLICEOSOMAL PROTEIN COMPLEXED WITH AN RNA HAIRPIN
    OUBRIDGE, C
    ITO, N
    EVANS, PR
    TEO, CH
    NAGAI, K
    [J]. NATURE, 1994, 372 (6505) : 432 - 438
  • [30] A SPECIFIC TRANSCRIPTION FACTOR THAT CAN BIND EITHER THE 5S RNA GENE OR 5S RNA
    PELHAM, HRB
    BROWN, DD
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1980, 77 (07): : 4170 - 4174