Isolation and Partial Characterization of Novel, Structurally Uniform (Hfq6)n≥8 Assemblies Carrying Accessory Transcription and Translation Factors.

In growing E. coli cells, the transcription-translation complexes (TTCs) form characteristic foci; however, the exact molecular composition of these superstructures is not known with certainty. Herein, we report that, during our recently developed "fast" procedures for purification of E. coli RNA polymerase (RP), a fraction of the RP's α/RpoA subunits is displaced from the core RP complexes and copurifies with multiprotein superstructures carrying the nucleic acid-binding protein Hfq and the ribosomal protein S6. We show that the main components of these large multiprotein assemblies are fixed protein copy-number (Hfq6)n≥8 complexes; these complexes have a high level of structural uniformity and are distinctly unlike the previously described (Hfq6)n "head-to-tail" polymers. We describe purification of these novel, structurally uniform (Hfq6)n≥8 complexes to near homogeneity and show that they also contain small nonprotein molecules and accessory S6. We demonstrate that Hfq, S6, and RP have similar solubility profiles and present evidence pointing to a role of the Hfq C-termini in superstructure formation. Taken together, our data offer new insights into the composition of the macromolecular assemblies likely acting as scaffolds for transcription complexes and ribosomes during bacterial cells' active growth.

Protein complex immunological separation assay (ProCISA): a technique for investigating single protein properties

Analysis of the posttranslational modification of proteins, such as phosphorylation,might yield misleading results due to the presence of other proteins with similar electrophoreticproperties that coimmunoprecipitate with the target protein. The aim ofthe present work was to develop a reliable, easy and economical technique to completelyisolate a protein from its complex. Here we present a new assay developed tofully isolate proteins from macromolecular complexes that consists of an initialSDS/PAGE (under reducing conditions), which isolates the target protein, followedby transfer of the proteins to a buffer, from which the target protein is recaptured byconventional immunoprecipitation. This technique, that we have termed “ProteinComplex Immunological Separation Assay” (ProCISA), successfully separated proteinsof different sizes, such as pp60Src and the IP3 receptor (IP3R), from their complexes.We show that ProCISA allows the investigation of the tyrosine phosphorylationstate of isolated proteins. This technique could also be used to study other posttranslationalmodifications without risk of misleading results resulting from contaminationwith other proteins of similar electrophoretic mobility which complex with theprotein of interest (AU)

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