Specific cross-linking of proteins S7 and L4 to ribosomal RNA, by UV irradiation of Escherichia coli ribosomal subunits

Abstract

Radioactive 30S and 50S subunits from E. coli ribosomes were irradiated with UV light, under conditions giving rise to approximately 10% cross-linking of protein to ribosomal RNA. Irradiation to levels of cross-linking higher than 10% caused unfolding of the ribosomal subunits, even in the presence of 5 mM magnesium. The specificity of the cross-linking reaction at this low level was found to be extremely high. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease and then analysed on Sarkosyl gels. S7 was found to be the primary target of the cross-linking reaction in the 30S particle. This was proven by using subunits from both E. coli MRE 600 and A19, whose respective S7 species differ markedly. In the 50S particle, L4 was the primary target, although L2 was also cross-linked to a small extent. Ambiguity in the identification of L4 in the Sarkosyl system was resolved by two-dimensional electrophoresis, which was also used to demonstrate a genuine linkage to RNA in the case of both S7 and L4; proteins spots containing 32P were observed, derived from these two proteins, when subunits containing 32P-RNA were irradiated, treated with nuclease, and applied to the electrophoresis. The identities of S7, L4 and L2 were finally confirmed by Ouchterlony tests with protein-specific anti-sera.

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

“…For analysis of the percentage of RNA-protein cross-linking, reaction mixtures containing about 2 A260 units of radioactive subunits were submitted to electrophoresis on 3 % polyacrylamide gels containing 0.1 sodium dodecyl sulphate, exactly as described elsewhere [13].…”

“…Reaction mixtures containing approximately 3 A260 units of radioactive subunits and 300 A260 units of unlabelled subunits were incubated with formaldehyde as above (0.03:d and O.lO%, for 3 0 3 and 50-S subunits, respectively), and the subunits were then precipitated with ethanol before applying to 7.5 -30 % sucrose gradients containing 0.1 % dodecyl sulphate, as described [13]. In this case however, the gradients were run in triethanolamine-HC1 buffer instead of Tris-HC1.…”

“…In this case however, the gradients were run in triethanolamine-HC1 buffer instead of Tris-HC1. After radioactive analysis of the gradients, fractions containing the cross-linked RNA-protein complex were concentrated by ethanol precipitation, treated with a mixture of ribonucleases A and T1 in sarkosyl buffer as described [13], and the proteins were then precipitated by addition of 4 vol. acetone, 1 vol.…”

“…We have recently published a partial localisation of the region of 16-S RNA which is cross-linked to protein S7 by ultraviolet irradiation [8], S7 being the only 30-S subunit protein which is covalently linked to RNA to any significant extent under the conditions used [13]. The next obvious development in this field is to determine whether the same type of information can be extracted from subunits in which many proteins are cross-linked simultaneously, since this is the situation which one would expect to arise from the use of most RNA-protein cross-linking procedures.…”

Eukaryotic elongation factor Tu is present in mRNA‐protein complexes

“…eEF-Tu from mRNP. Other investigators have found that UV crosslinking to RNA results in a slight increase in the apparent size of proteins even after RNase digestion [15,16]. On the basis of these data it seems likely that mRNA in rabbit reticulocytes is associated with eEF-Tu.…”