Localization of spermine binding sites in 23S rRNA by photoaffinity labeling: parsing the spermine contribution to ribosomal 50S subunit functions.
Polyamine binding to 23S rRNA was investigated, using a photoaffinity labeling approach. This was based on the covalent binding of a photoreactive analog of spermine, N1-azidobenzamidino (ABA)-spermine, to Escherichia coli ribosomes or naked 23S rRNA under mild irradiation conditions. The cross-linking sites of ABA-spermine in 23S rRNA were determined by RNase H digestion and primer-extension analysis. Domains I, II, IV and V in naked 23S rRNA were identified as discrete regions of preferred cross-linking. When 50S ribosomal subunits were targeted, the interaction of the photoprobe with the above 23S rRNA domains was elevated, except for helix H38 in domain II whose susceptibility to cross-linking was greatly reduced. In addition, cross-linking sites were identified in domains III and VI. Association of 30S with 50S subunits, poly(U), tRNA(Phe) and AcPhe-tRNA to form a post-translocation complex further altered the cross-linking, in particular to helices H11-H13, H21, H63, H80, H84, H90 and H97. Poly(U)-programmed 70S ribosomes, reconstituted from photolabeled 50S subunits and untreated 30S subunits, bound AcPhe-tRNA in a similar fashion to native ribosomes. However, they exhibited higher reactivity toward puromycin and enhanced tRNA-translocation efficiency. These results suggest an essential role for polyamines in the structural and functional integrity of the large ribosomal subunit.
Xaplanteri
Maria A
MA
Laboratory of Biochemistry, School of Medicine, University of Patras GR-26500 Patras, Greece.
Petropoulos
Alexandros D
AD
Dinos
George P
GP
Kalpaxis
Dimitrios L
DL
D001386:Azides / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010458:Peptidyl Transferases / Q000378:metabolism; D019920:Photoaffinity Labels; D011072:Poly U / Q000378:metabolism; D014176:Protein Biosynthesis; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism; D013096:Spermine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism
D001386:Azides; D019920:Photoaffinity Labels; D012338:RNA, Ribosomal, 23S; D012346:RNA, Transfer, Amino Acyl; C006383:tRNA, N-acetylphenylalanine-; C068638:N(1)-(azidobenzamidino)spermine; D011072:Poly U; D013096:Spermine; D010458:Peptidyl Transferases