[Escherichia coli ribosomes as the model to test new photoactivated tRNA analogues, containing 6-thioguanosine]

Abstract

Photoreactive derivatives of tRNAs, containing 6-thioguanosine or diazirine derivative of 5-methyleneaminouridine were compared as probes to modify Escherichia coli ribosomes. The derivatives of tRNA were synthesized by T7 transcription Proportion of the modified nucleotide analogues was optimised to obtain good yield, analogue incorporation and binding to the ribosome. Complexes of the tRNA analogues with the ribosomal P-site were irradiated with mild UV light. Cross-links were analysed by oligonucleotide-directed hydrolysis of rRNA by RNase H and reverse transcription. 6-thioguanosine was proved to be a perspective reagent for cross-linking studies of complex ribonucleoproteides.