Distance Measurement by Energy Transfer: The 3′ End of 16‐S RNA and Proteins S4 and S17 of the Ribosome of Escherichia coli

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Abstract
Escherichia coli ribosomal proteins S4 and S17 were specifically labelled at their thiol groups with the acetylaminoethyl‐dansyl and/or bimane fluorophores. Each formed a complex with 16‐S RNA and, when the other 30‐S ribosomal proteins were added, a complete 30‐S subunit with at least partial activity. If the 3′ end of the RNA was also labelled (with fluorescein) then the distance between the two fluorophores could be measured by Förster‐type energy transfer. The result for S4 was 6.0 nm (60 Å) in the ribonucleoprotein complex and 5.6 nm (56 Å) in the 30‐S subunit, and for S17 6.3 nm (63 Å) in the complex and 6.2 nm (62 Å) in the subunit. There is no evidence for a major change in the relative disposition of the 3′ and 5′ ends of the 16‐S RNA during formation of the 30‐S subunit. Sources of error are discussed, including the question of multiple labelling.In order to measure more accurately the extent of energy transfer a procedure based upon enzymic digestion was developed and is detailed in this paper.