The Use of Formaldehyde in RNA‐Protein Cross‐linking Studies with Ribosomal Subunits from Escherichia coli

Abstract

Radioactive subunits from Escherichia coli ribosomes were treated with formaldehyde, with a view to inducing a controlled cross‐linking reaction between protein and ribosomal RNA. Under the conditions described, about 10–15% of the total protein remained bound to the RNA in the presence of dodecyl sulphate, and little or no irreversible protein‐protein cross‐linking was observed. The RNA‐protein complexes are of a rather unstable nature, and the reaction is spontaneously reversible.The proteins involved in the cross‐linked complexes were examined by polyacrylamide gel electrophoresis, after removal of unbound protein and nuclease treatment to digest the RNA‐protein complex. This showed that the individual ribosomal proteins were cross‐linked to varying degrees, and most of the proteins were involved in the reaction at least to some extent. A distinct group of proteins from each subunit were, however, reproducibly cross‐linked in high yield.The formaldehyde‐treated subunits were subjected to a controlled hydrolysis with ribonuclease T1, and RNA fragments of known nucleotide sequence containing the cross‐linked proteins were isolated. Despite the reversibility of the cross‐linking reaction, sufficient cross‐linked protein survived the experiment to enable an analysis to be made of proteins associated with the various RNA fragments.In the 50‐S subunit, proteins L27 and L32 were found to be cross‐linked to the 18‐S RNA fragment which arises from the 3′ end of 23‐S RNA. L2, L6 and L16 were also found in this fragment in small amounts. In contrast, L13 appeared to be linked to both the 18‐S RNA and the 13‐S RNA fragment from the 5′ end of 23‐S RNA. In the 30‐S subunit, proteins S3, S5, S9 (11), S13, and to some extent S4, S7, S12 and S18, were found associated with an RNA region comprising approximately 900 nucleotides at the 5′ end of 16‐S RNA. S4, S9 (11), S13, and to a lesser extent S7 and S14, were found in an RNA region comprising about 450 nucleotides near the 3′ end of 16‐S RNA, but lacking the 3′‐terminal 150 nucleotides. Protein S21 was cross‐linked to 16‐S RNA, but was not found in either of the two RNA regions above.