The identification of spermine binding sites in 16S rRNA allows interpretation of the spermine effect on ribosomal 30S subunit functions.
A photoreactive analogue of spermine, N1-azidobenzamidino (ABA)-spermine, was covalently attached after irradiation to Escherichia coli 30S ribosomal subunits or naked 16S rRNA. By means of RNase H digestion and primer extension, the cross-linking sites of ABA-spermine in naked 16S rRNA were characterised and compared with those identified in 30S subunits. The 5' domain, the internal and terminal loops of helix H24, as well as the upper part of helix H44 in naked 16S rRNA, were found to be preferable binding sites for polyamines. Association of 16S rRNA with ribosomal proteins facilitated its interaction with photoprobe, except for 530 stem-loop nt, whose modification by ABA-spermine was abolished. Association of 30S with 50S subunits, poly(U) and AcPhe-tRNA (complex C) further altered the susceptibility of ABA-spermine cross-linking to 16S rRNA. Complex C, modified in its 30S subunit by ABA-spermine, reacted with puromycin similarly to non-photolabelled complex. On the contrary, poly(U)-programmed 70S ribosomes reconstituted from photolabelled 30S subunits and untreated 50S subunits bound AcPhe-tRNA more efficiently than untreated ribosomes, but were less able to recognise and reject near cognate aminoacyl-tRNA. The above can be interpreted in terms of conformational changes in 16S rRNA, induced by the incorporation of ABA-spermine.
Amarantos
Ioannis
I
Laboratory of Biochemistry and Laboratory of Biology, School of Medicine, University of Patras, GR-26500 Patras, Greece.
Zarkadis
Ioannis K
IK
Kalpaxis
Dimitrios L
DL
D001386:Azides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D003432:Cross-Linking Reagents; D007700:Kinetics; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D013096:Spermine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D014316:Tritium
D001386:Azides; D003432:Cross-Linking Reagents; D012336:RNA, Ribosomal, 16S; D012346:RNA, Transfer, Amino Acyl; D012360:RNA, Transfer, Phe; D014316:Tritium; C068638:N(1)-(azidobenzamidino)spermine; D013096:Spermine; D016914:Ribonuclease H