Localization of spermine binding sites in 23S rRNA by photoaffinity labeling: parsing the spermine contribution to ribosomal 50S subunit functions
| Autor: | Alexandros D. Petropoulos, Maria A. Xaplanteri, Dimitrios L. Kalpaxis, George P. Dinos |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Poly U
Azides Binding Sites Base Sequence Photoaffinity labeling Eukaryotic Large Ribosomal Subunit Molecular Sequence Data Photoaffinity Labels RNA Transfer Amino Acyl Ribosomal RNA Biology Polyamine binding Article RNA Ribosomal 23S Biochemistry Ribosomal protein 23S ribosomal RNA Protein Biosynthesis Large ribosomal subunit Peptidyl Transferases Escherichia coli Genetics Spermine Eukaryotic Ribosome Ribosomes |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gki557 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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