Structural insight of human DEAD-box protein rck/p54 into its substrate recognition with conformational changes.
Human rck/p54, a product of the gene cloned at the breakpoint of t(11; 14) (q23;q32) chromosomal translocation on 11q23 in B-cell lymphoma, is a member of the DEAD-box RNA helicase family. Here, the crystal structure of Nc-rck/p54, the N-terminal core domain of rck/p54, revealed that the P-loop in motif I formed a closed conformation, which was induced by Asn131, a residue unique to the RCK subfamily. It appears that ATP does not bind to the P-loop. The results of dynamic light scattering revealed to ATP-induced conformational change of rck/p54. It was demonstrated that free rck/p54 is a distended molecule in solution, and that the approach between N-terminal core and C-terminal domains for ATP binding would be essential when unwinding RNA. The results from helicase assay using electron micrograph, ATP hydrolytic and luciferase assay showed that c-myc IRES RNA, whose secondary structure regulates IRES-dependant translation, was unwound by rck/p54 and indicated that it is a good substrate for rck/p54. Over-expression of rck/p54 in HeLa cells caused growth inhibition and cell cycle arrest at G2/M with down-regulation of c-myc expression. These findings altogether suggest that rck/p54 may affect the IRES-dependent translation of c-myc even in the cells.
Matsui
Tsutomu
T
Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta-cho 4259, Midori-ku, Yokohama, 226-8501, Japan.
Hogetsu
Keita
K
Usukura
Jiro
J
Sato
Takao
T
Kumasaka
Takashi
T
Akao
Yukihiro
Y
Tanaka
Nobuo
N
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000235:genetics / Q000502:physiology; D049109:Cell Proliferation; D053487:DEAD-box RNA Helicases; D015536:Down-Regulation; D016195:G2 Phase / Q000235:genetics / Q000502:physiology; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D012313:RNA; D000255:Adenosine Triphosphate; D012316:RNA Nucleotidyltransferases; C094683:DDX6 protein, human; D053487:DEAD-box RNA Helicases