Add time:08/08/2019 Source:sciencedirect.com
In E. coli, a single oligomeric enzyme transcribes the genomic DNA, while multiple auxiliary proteins and regulatory RNA interact with the core RNA polymerase (RP) during different stages of the transcription cycle to influence its function. In this work, using fast protein isolation techniques combined with mass spectrometry (MS) and immuno-analyses, we studied growth phase-specific changes in the composition of E. coli transcription complexes. We show that RP isolated from actively growing cells is represented by prevalent double copy assemblies and single copy RP–RNA and RP–RNA–RapA complexes. We demonstrate that RpoD/σ70 obtained in fast purification protocols carries tightly associated RNA and show evidence pointing to a role of sigma-associated RNA in the formation of native RP–(RNA)–RpoD/σ70 (holoenzyme) complexes. We report that enzymes linked functionally to the metabolism of lipopolysaccharides co-purify with RP–RNA complexes and describe two classes of RP-associated molecules (phospholipids and putative phospholipid–rNT species). We hypothesize that these modifications could enable anchoring of RP–RNA and RNA in cell membranes. We also report that proteins loosely associated with ribosomes and degradosomes (S1, Hfq) co-purify with RP–RNA complexes isolated from actively growing cells – a result consistent with their proposed roles as adaptor-proteins. In contrast, GroEL, SecB, and SecA co-purified with RP obtained from cells harvested in early stationary phase. Our results demonstrate that fast, affinity chromatography-based isolation of large multi-protein assemblies in combination with MS can be used as a tool for analysis of their composition and the profiling of small protein-associated molecules (SPAM).
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