Crosslinks between ribosomal proteins of 30S subunits in 70S tight couples and in 30S subunits

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Abstract
Escherichia coli ribosome 70S tight couples and 30S subunits derived from them were modified with 2-iminothiolane under conditions where .apprx. 2 SH groups/protein were added to the ribosomal particles. The 70S and 30S particles were not treated with elevated concentrations of NH4Cl, in contrast to those used in earlier studies. The modified particles were oxidized to promote disulfide bond formation. Proteins were extracted from the cross-linked particles using conditions to prelude disulfide interchange. Disulfide-link protein complexes were fractionated on the basis of charge by electrophoresis in polyacrylamide/urea gels at pH 5.5. The proteins from sequential slices of the urea gels were analyzed by 2-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Final identification of proteins in cross-linked complexes was made by radioiodination of the proteins, followed by 2-dimensional polyacrylamide/urea gel electrophoresis. Attention was focused on cross-links between 30S proteins. The identification of 27 cross-linked dimers and 2 trimers of 30S proteins, all but 1 of which were found in both 70S ribosomes and free 30S subunits in similar yields, is reported. Seven of the cross-links, S3-S13, S13-S21, S14-S19, S7-S12, S9-S13, S11-S21 and S6-S18-S21, were not reported previously when 2-iminothiolane was used. Cross-links S3-S13, S13-S21, S7-S12, S11-S21 and S6-S18-S21 are reported for the 1st time. The identification of the 7 new cross-links is illustrated and discussed in detail. Ten of the dimers reported in earlier studies using 30S subunits treated with high salt concentrations were not found in the experiments reported here.

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