Biochemical & Biophysical Studies of an Hfq Homolog from a Deep-branching Bacterium 660 views

RNA-based regulation enables exquisite control over the extent and timing of gene expression, enabling bacteria to rapidly respond to their environment. The bacterial host factor Hfq is involved in regulation of gene expression via its role as an RNA chaperone; Hfq binds to sRNAs and regulates their stability, and also interacts with sRNA and mRNA to facilitate their annealing. Hfq has been shown to preferentially bind adenosine– and uracil–rich RNAs. Here, we discovered that the Hfq from Thermotoga maritima (Tma) interacts with short uracil/cytosine-rich nanoRNAs when recombinantly expressed in Escherichia coli. These Hfq-binding nanoRNAs interact with nanomolar affinities and feature 5’-monophosphate and 3’-hydroxyl end chemistries. Previous work by others revealed the functional form of Hfq in Escherichia coli and other bacterial species is a homohexamer. Biophysical and biochemical characterization of Tma Hfq supports the formation of both homohexamers and homododecamers. Isothermal calorimetry and semi-native Western blot analysis indicate that the hexameric and dodecameric states are in equilibrium. Fluorescence polarization assays show that both oligomeric states bind to adenosine- and uracil-rich RNAs with nanomolar affinities. To elucidate the atomic basis of Tma Hfq function, we determined the crystal structure of this protein to 2.1 Å resolution, which further demonstrated that Tma Hfq forms a homohexamer. I also describe initial in vivo studies of the expression levels, cellular localization, and RNA binding partners of Hfq in Tma. Polyclonal antibodies to Tma Hfq were propagated, purified and characterized for downstream enrichment and detection assays. A quantitative Western blot was developed to determine the concentration of Tma Hfq in cell lysate; the concentrations determined via this assay, along with cell counts determined by flow cytometry, are being used to determine the expression level of Tma Hfq at different phases in the growth of this bacterium. Initial images of immunogold-labeled Tma cells have been collected by electron microscopy to identify and localize Hfq in vivo. Most recently, co-immunoprecipitation assays are being pursued to isolate Hfq along with its native binding partners in Tma; downstream analyses of these data include identification of protein and RNA binding partners via mass spectrometry-based proteomics and next-generation sequencing.

PHD (Doctor of Philosophy)

Hfq; RNA-binding; oligomerization; crystal structure; Thermotoga maritima; nanoRNA

English

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2014-04-22