RNA-protein cross-linking in Escherichia coli 50S ribosomal subunits; determination of sites on 23S RNA that are cross-linked to proteins L2, L4, L24 and L27 by treatment with 2-iminothiolane.
RNA-protein cross-links were introduced into E. coli 50S ribosomal subunits by treatment with 2-iminothiolane followed by mild ultraviolet irradiation. After partial digestion of the RNA, the cross-linked RNA-protein complexes were separated by our recently published three-step procedure. In cases where this separation was inadequate, a further purification step was introduced, involving affinity chromatography with antibodies to the ribosomal 50S proteins. Analysis of the isolated complexes enabled four new cross-link sites on the 23S RNA to be identified, as well as re-confirming several previously established sites. The new sites are as follows: Protein L2 is cross-linked within an oligonucleotide at positions 1818-1823 in the 23S RNA, protein L4 within positions 320-325, protein L24 within positions 99-107, and protein L27 within positions 2320-2323.
Gulle
H
H
Max-Plank-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin-Dahlem, FRG.
Hoppe
E
E
Osswald
M
M
Greuer
B
B
Brimacombe
R
R
Stöffler
G
G
D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004926:Escherichia coli / Q000032:analysis*; D007096:Imidoesters / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012335:RNA, Ribosomal / Q000378:metabolism*; D012339:RNA, Ribosomal, 28S / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000528:radiation effects / Q000648:ultrastructure*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D007096:Imidoesters; D012335:RNA, Ribosomal; D012339:RNA, Ribosomal, 28S; D012269:Ribosomal Proteins; C005905:methyl 4-mercaptobutyrimidate