Contact sites of peptide-oligoribonucleotide cross-links identified by a combination of peptide and nucleotide sequencing with MALDI MS.
We have investigated peptide-oligoribonucleotide complexes isolated from cross-linked Escherichia coli 30S ribosomal subunits in order to identify the contact sites of these complexes at the molecular level. For this purpose, reversed-phase (RP) HPLC-purified peptide-oligoribonucleotide complexes were submitted to N-terminal amino acid sequencing in order to determine the cross-linked peptide moiety and were analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for calculation of the nucleotide composition of the cross-linked complex. Subsequently, for nucleotide sequence information the complexes were partially hydrolyzed or treated with exonucleases and analyzed again by MALDI-MS. Applying this technique, we were able to identify the cross-linked oligoribonucleotide parts in contact with distinct peptide regions derived from ribosomal proteins S4, S7, S8, and S17 from E. coli.
Urlaub
H
H
Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany.
Thiede
B
B
Müller
E C
EC
Wittmann-Liebold
B
B
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000032:analysis* / Q000378:metabolism; D012335:RNA, Ribosomal / Q000032:analysis* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000032:analysis* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C040469:ribosomal protein S30