Identification and Sequence Analysis of RNA-Protein Contact Sites by N-Terminal Sequencing and MALDI-MS

Crosslinking techniques have been used widely to obtain meaningful structural information on RNA-protein interactions. Exact data on the contact sites of RNA-protein complexes at the molecular level are required for a detailed modeling of three-dimensional structures within the complexes and for the identification of recognition motifs. For this purpose an approach was developed to determine the contact sites of RNA-protein crosslinks by combining N-terminal sequencing and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This approach allowed the precise localization of the contact sites within the complex ribonucleoprotein particle of the native 30S ribosomal subunit ( 1 – 4 ). Crosslinking experiments are performed either by ultraviolet (UV)-irradiation or by combining 2-iminothiolane and UV treatment. The generated RNA-protein crosslinks are digested with ribonuclease T ₁ and endoprotease (Lys-C or Glu-C) and purified by size-exclusion chromatography and reverse phase-high-performance liquid chromatography (RP-HPLC). The purified oligoribonucleotide-peptide complexes are then subjected to N-terminal sequencing and MALDI-MS to identify the position of the crosslink in the protein and RNA (Fig. 1 ). The amino acid sequence of the peptide enabled the identification of the corresponding ribosomal protein and a gap in the sequence defined the crosslinking position. The mass spectrometrical analysis of the complexes prior to and after partial alkaline hydrolysis and treatment with 5′→3′ phosphodiesterase led to the identification of the composition,

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