Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors--the spliceosome--is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.

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The chemical steps of bacteriophage Mu DNA transposition take place within a higher order nucleoprotein structure. We describe a novel intermediate that precedes the previously characterized transpososomes and directly demonstrates the interaction of a distant enhancer element with recombination regions. The transpositional enhancer interacts with the Mu left and right ends to form a three-site synaptic (LER) complex. Under normal reaction conditions, the LER complex is rapidly converted into the more stable Mu transpososomes. However, mutation of the Mu terminal nucleotides results in accumulation of the LER and a failure to form the type 0 transpososome. During the transition from LER to type 0, the Mu DNA termini and the active site of the transposase engage in a catalytically competent conformation.

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