ABSTRACT
The uptake of zinc, which is vital in trace amounts, is tightly controlled in bacteria. For this control, bacteria of the Streptococcaceae group use a Zn(II)-binding repressor named ZitR in lactococci and AdcR in streptococci, while other bacteria use a Zur protein of the Ferric uptake regulator (Fur) superfamily. ZitR and AdcR proteins, characterized by a winged helix-turn-helix DNA-binding domain, belong to the multiple antibiotic resistance (MarR) superfamily, where they form a specific group of metallo-regulators. Here, one such Zn(II)-responsive repressor, ZitR of Lactococcus lactis subspecies cremoris strain MG1363, is characterized. Size Exclusion Chromatography-coupled to Multi Angle Light Scattering, Circular Dichroism and Isothermal Titration Calorimetry show that purified ZitR is a stable dimer complexed to Zn(II), which is able to bind its two palindromic operator sites on DNA fragments. The crystal structure of ZitR holo-form (Zn(II)4-ZitR2), has been determined at 2.8 Å resolution. ZitR is the fourth member of the MarR metallo-regulator subgroup whose structure has been determined. The folding of ZitR/AdcR metallo-proteins is highly conserved between both subspecies (cremoris or lactis) in the Lactococcus lactis species and between species (Lactococcus lactis and Streptococcus pneumoniae or pyogenes) in the Streptococcaceae group. It is also similar to the folding of other MarR members, especially in the DNA-binding domain. Our study contributes to better understand the biochemical and structural properties of metallo-regulators in the MarR superfamily.
Subject(s)
Bacterial Proteins/metabolism , Lactococcus lactis/metabolism , Repressor Proteins/metabolism , Zinc/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Binding Sites , Crystallography, X-Ray , Lactococcus lactis/chemistry , Models, Molecular , Protein Binding , Protein Conformation , Protein Multimerization , Repressor Proteins/chemistryABSTRACT
RRP6 is a 3'-5' exoribonuclease associated to the eukaryotic exosome, a multiprotein complex essential for various RNA processing and degradation pathways. In Trypanosoma brucei, RRP6 associates with the exosome in stoichiometric amounts and was localized in both cytoplasm and nucleus, in contrast to yeast Rrp6 which is exclusively nuclear. Here we report the biochemical and structural characterization of T. brucei RRP6 (TbRRP6) and its interaction with the so-called T. brucei Exosome Associated Protein 3 (TbEAP3), a potential orthologue of the yeast Rrp6 interacting protein, Rrp47. Recombinant TbEAP3 is a thermo stable homodimer in solution, however it forms a heterodimeric complex with TbRRP6 with 1â¶1 stoichiometry. The crystallographic structure of the TbRRP6 catalytic core exposes for the first time the native catalytic site of this RNase and also reveals a disulfide bond linking two helices of the HRDC domain. RNA degradation assays show the distributive exoribonuclease activity of TbRRP6 and novel findings regarding the structural range of its RNA substrates. TbRRP6 was able to degrade single and double-stranded RNAs and also RNA substrates containing stem-loops including those with 3' stem-loop lacking single-stranded extensions. Finally, association with TbEAP3 did not significantly interfere with the TbRRP6 catalytic activity in vitro.
