Identification and sequence analysis of contact sites between ribosomal proteins and rRNA in Escherichia coli 30 S subunits by a new approach using matrix-assisted laser desorption/ionization-mass spectrometry combined with N-terminal microsequencing.
Cross-linked peptide-oligoribonucleotide complexes derived from distinct regions of the rRNA and individual ribosomal proteins of the 30 S ribosomal subunits from Escherichia coli were isolated and purified. Cross-linking sites at the amino acid and nucleotide level were determined by N-terminal amino acid sequence analysis in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). MALDI-MS analysis performed subsequent to a partial alkaline hydrolysis of cross-linked peptide-oligoribonucleotide complexes allowed for the first time the cross-linked rRNA moiety to be sequenced by this technique. In this manner Lys-44 in S4 was determined to be cross-linked to the oligoribonucleotide at positions 1531-1542 on the 16 S RNA (whereby either U-1541 or A-1542 is the actual cross-link site), Lys-75 in S7 to positions 1374-1379 (C-1378 cross-linked), Met-114 in S7 to 1234-1241 (U-1240 cross-linked), Lys-55 in S8 to 651-654 (U-653 cross-linked), and Lys-29 in S17 to 629-633 (U-632 cross-linked). The novel approach applied here promises to be useful for similar studies on other known protein.RNA complexes.
Urlaub
H
H
Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Strabetae 10, D-13125 Berlin, Germany.
Thiede
B
B
Müller
E C
EC
Brimacombe
R
R
Wittmann-Liebold
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000378:metabolism*; D008239:Lysine; D008715:Methionine; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003432:Cross-Linking Reagents; D009843:Oligoribonucleotides; D010446:Peptide Fragments; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; C037084:ribosomal protein S17; D008715:Methionine; D008239:Lysine