The environment of 5S rRNA in the ribosome: cross-links to 23S rRNA from sites within helices II and III of the 5S molecule

Three contiguous fragments of Escherichia coli 5S rRNA were prepared by T7 transcription from synthetic DNA templates. The central fragment, comprising residues 33-71 of the molecule, was transcribed in the presence of 4-thiouridine triphosphate together with [(32)P]UTP. The three transcripts were ligated together, yielding a 5S rRNA analogue carrying 4-thiouridine residues at positions 40, 48, 55 and 65 in helices II and IH. After ligation, the 4-thiouridine residues were derivatised with p-azidophenacyl bromide. The modified 5S rRNA was reconstituted into 50S subunits and these subunits were used to prepare 70S ribosomes in the presence or absence of tRNA and mRNA, The azidophenyl groups were then photoactivated by mild irradiation at 300 nm and the products of cross-linking analysed by our standard procedures. Multiple crosslinks from 5S rRNA to two distinct regions of the 23S rRNA were observed. The first region was located in helix 38 in Domain II of the 23S molecule, with cross-links at sites between nucleotides 885 and 922. The second region covered helices 81-85 in Domain V, with sites between nucleotides 2272 and 2345, Taken together with previous data, these results serve to define the arrangement of the 5S rRNA molecule relative to the 23S rRNA within the 50S subunit.