Hybridization selection of nucleic acid-protein complexes. 1. Detection of proteins cross-linked to specific mRNAs and DNA sequences by irradiation of intact Escherichia coli cells with ultraviolet light.
A method is presented for the detection in crude lysates of subnanogram amounts of proteins covalently bound to a specific nucleic acid sequence. The sensitivity of this method enabled us to study proteins cross-linked to specific DNA and mRNA sequences by irradiation of intact Escherichia coli cells with ultraviolet light. Among the proteins cross-linked to pBR322 DNA, the single strand binding protein, the HU-proteins, and the RNA polymerase beta and sigma subunits were present. Some, but not all proteins were cross-linked to 5-bromodeoxyuridine-substituted DNA more efficiently than to normal DNA. Ribosomal protein S1 is by far the most prominent protein cross-linked to mRNAs. Among the proteins cross-linked in smaller amounts to mRNAs are translation initiation factor IF 1, and at least six proteins of the 30 S ribosomal subunit, among which is S21. No 50 S proteins, nor IF-2, IF-3 or any of the elongation factors could be detected. Some UV-induced nucleic acid-protein cross-links were found to be heat-labile. It is concluded that the method employed may be used to compare the proteins interacting with different mRNAs, as well as single-copy DNA sequences from bacteria and eucaryotes with low complexity genomes.
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000528:radiation effects*; D015977:Eukaryotic Initiation Factor-1; D008722:Methods; D039861:Prokaryotic Initiation Factor-1; D011506:Proteins / Q000378:metabolism* / Q000528:radiation effects; D012333:RNA, Messenger / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D015977:Eukaryotic Initiation Factor-1; D039861:Prokaryotic Initiation Factor-1; D011506:Proteins; D012333:RNA, Messenger; D012269:Ribosomal Proteins