Journal Article

Localization of spermine binding sites in 23S rRNA by photoaffinity labeling: parsing the spermine contribution to ribosomal 50S subunit functions

Maria A. Xaplanteri, Alexandros D. Petropoulos, George P. Dinos and Dimitrios L. Kalpaxis

in Nucleic Acids Research

Volume 33, issue 9, pages 2792-2805
Published in print January 2005 | ISSN: 0305-1048
Published online January 2005 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/gki557

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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), tRNAPhe 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.

Journal Article.  9885 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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