Loop IV of 5S ribosomal RNA has contacts both to domain II and to domain V of the 23S RNA.

An analogue of 5S rRNA, containing a random distribution of thiouridine residues in place of normal uridine, was prepared by T7 transcription from a suitable DNA template. The modified RNA molecule was reconstituted into 50S or 70S ribosomes, and the thiouridine residues were activated by irradiation at 350 nm. Crosslinks generated between the 5S and 23S RNA were analyzed by our standard procedures. Two crosslink sites were identified, one to residue A-960 at the loop-end of helix 39 in Domain II, and the other to C-2475 at the loop-end of helix 89 in Domain V of the 23S RNA. Both crosslinks involved residue U-89 of the 5S RNA, that in Domain V corresponding to the principal crosslink found in a previously published series of experiments. The relative intensities of the two crosslink sites were found to be highly dependent on individual preparations of 50S ribosomal proteins and 23S RNA. The results are discussed in terms of the three-dimensional folding and dynamics of the 23S RNA within the 50S subunit.