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 TI, 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 (ll), S 13, and to some extent S4, S7, S 12 and S 18, were found associated with an RNA region comprising approximately 900 nucleotides at the 5' end of 16-S RNA. S4, S9 (ll), S13, and to a lesser extent S 7 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.Two different biochemical approaches are currently being used to investigate RNA-protein interactions in the Escherichia coli ribosome. The first of these involves the study of complexes between isolated proteins and ribosomal RNA or fragments of ribosomal , and this method is basically intended to discover those regions of the RNA which contain stable 'binding sites' for individual proteins. The second approach involves the isolation of ribonucleoprotein fragments from ribosomal subunits, the purpose here being to investigate topographical relationships 'between proteins or groups

Electron microscopic determination of the binding sites of ribosomal proteins S4 and S8 on 16S RNA.

“…6C). This binding site has not been proposed by previous authors; however, several pieces of evidence from biochemical experiments strengthen our suggestion that this is a specific binding site for S4 (8,20,21 As mentioned above, the minimum size of the loops is 0.09 ,gm (250 bases). This loop size localizes the second binding region to at least 945 bases (250 bases plus the length of the free end, 695 bases) from the 3' end of 16S RNA and at least 250 bases from the binding site at the 5' end.…”

An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues

“…Moreover, formaldehyde (HCHO) is the main constituent of formalin that is responsible for the cross-linkage between proteins and DNA or RNA, which in turn could be limiting for further use of the nucleic acids (e.g. Zsikla et al, 2004;Moller et al, 1977). Finally, one of the recent methods described to remove formaldehyde from the specimens is the application of graded ethanol washes followed by a drying critical point (Fang et al, 2002).…”

Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures.

“…In a different set of approaches, DNA-bound proteins are first crosslinked to DNA by treatments with UV light (6), formaldehyde (HCHO) (7), dimethyl sulfate (8), or a variety of other agents (9). Some of the above methods, in particular DNA modification by dimethyl sulfate (10,11) On the other hand, HCHO produces DNA-protein crosslinks both in vitro and in vivo (7,(13)(14)(15)(16)(17)(18) and at the same time displays virtually no reactivity toward free double-stranded DNA (19,20). Since HCHO produces DNA-protein (7,(13)(14)(15)(16), , and protein-protein (18) crosslinks, its addition to living cells results within minutes in formation of crosslinked networks of biopolymers, thus preventing any large-scale redistribution of cellular components upon prolonged ("limit") fixation.…”