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 fr om cross-linked Escherichia coli 30S ribosomal subunits in order to id entify the contact sites of these complexes at the molecular level. Fo r this purpose, reversed-phase (RP) HPLC-purified peptide-oligoribonuc leotide complexes were submitted to N-terminal amino acid sequencing i n 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-l inked complex. Subsequently, for nucleotide sequence information the c omplexes were partially hydrolyzed or treated with exonucleases and an alyzed again by MALDI-MS. Applying this technique, we were able to ide ntify the cross-linked oligoribonucleotide parts in contact with disti nct peptide regions derived from ribosomal proteins S4, S7, S8, and S1 7 from E. coli.