Use of aryl azide cross-linkers to investigate protein-protein interactions: an optimization of important conditions as applied to Escherichia coli RNA polymerase and localization of a sigma 70-alpha cross-link to the C-terminal region of alpha.
In an effort to better understand protein-protein photoaffinity cross-linking using aryl azides, we have tested a number of factors influencing the cross-linking of the sigma 70 subunit of Escherichia coli RNA polymerase to core RNA polymerase. These factors include the effect of the incubations necessary for the derivatization of the protein on enzyme activity, the effect of overhead lighting on azide stability, the effect of reducing agents on azide stability, aggregation of the derivatized protein, and a comparison of two types of aryl azide cross-linkers, N-[(5-azido-2-nitrobenzoyl)oxy]succinimide (ANB-NOS) and (N-hydroxysuccinimidyl)-4-azidosalicylic acid (NHS-ASA). We found that derivatization proceeds effectively in a buffer similar to the buffer used during protein purification, that overderivatization can cause protein aggregation, that room lighting does not appreciably destroy aryl azides, and that 0.1 mM DTT is a better choice of reducing agent than 5 mM 2-mercaptoethanol. The cross-link products were separated by SDS gel electrophoresis and identified on Western blots by cross-reactivity with monoclonal antibodies to the individual subunits of RNA polymerase. In agreement with previous work (Coggins et al., 1977; Hillel & Wu, 1977), it was possible to cross-link sigma 70 to all three of the subunits of RNA polymerase. With a combination of gel analysis, chemical cleavage, and immunodetection, it is possible to demonstrate that sigma 70 cross-links to the alpha subunit between residues 209 and 329.
McMahan
S A
SA
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison 53706.
Burgess
R R
RR
D001386:Azides / Q000737:chemistry* / Q000378:metabolism; D001667:Binding, Competitive; D003432:Cross-Linking Reagents / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D015151:Immunoblotting; D008027:Light; D017830:Octoxynol; D011485:Protein Binding; D012459:Salicylates / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013388:Succinimides / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D001386:Azides; D003432:Cross-Linking Reagents; D012459:Salicylates; D013388:Succinimides; C049915:N-hydroxysuccinimidyl-5-azido-2-nitrobenzoate; D017830:Octoxynol; C046019:4-azidosalicylic acid N-hydroxysuccinimide ester; D012321:DNA-Directed RNA Polymerases