The environment of 5S rRNA in the ribosome: cross-links to the GTPase- associated area of 23S rRNA

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Abstract
Two photoreactive diazirine derivatives of uridine were used to study contacts between 5S rRNA and 23 rRNA in situ in Escherichia coli ribosomes. 2′-Amino-2′-deoxy-uridine or 5-methyleneaminouridine were introduced into 5S rRNA by T7 transcription. After incorporation of these uridine analogues into the transcript their amino groups were modified with 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl isothiocyanate or the N-hydroxysuccinimide ester of 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acid respectively. 5S rRNA carrying the photoreactive diazirine groups (referred to as the 2′-aminoribose derivative and the 5-methyleneamino derivative respectively) was reconstituted into 50S subunits or 70S ribosomes. After mild UV irradiation cross-links formed to 23S rRNA were analysed by standard procedures. All of the observed cross-links involved residue U89 of the 5S rRNA. Three nucleotides of 23S rRNA were cross-linked to this residue with the 5-methyleneamino derivative, namely U958, G1022 and G1138. With the 2′-aminoribose derivative a single cross-link was found, to U958. The significance of these cross-links for our understanding of the structure and function of 5S rRNA and its environment in the ribosome are discussed.

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