Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. Here, we identify a new copper-specific repressor (CsoR) of a copper-sensitive operon (cso) in Mycobacterium tuberculosis (Mtb) that is representative of a large, previously uncharacterized family of proteins (DUF156). Electronic and X-ray absorption spectroscopies reveal that CsoR binds a single-monomer mole equivalent of Cu(I) to form a trigonally coordinated (S(2)N) Cu(I) complex. The 2.6-A crystal structure of copper-loaded CsoR shows a homodimeric antiparallel four-helix bundle architecture that represents a novel DNA-binding fold. The Cu(I) is coordinated by Cys36, Cys65' and His61' in a subunit bridging site. Cu(I) binding negatively regulates the binding of CsoR to a DNA fragment encompassing the operator-promoter region of the Mtb cso operon; this results in derepression of the operon in Mtb and the heterologous host Mycobacterium smegmatis. Substitution of Cys36 or His61 with alanine abolishes Cu(I)- and CsoR-dependent regulation in vivo and in vitro. Potential roles of CsoR in Mtb pathogenesis are discussed.

She earned her M.S. degree in Chemistry in 2004 and her Ph.D. degree in Chemistry in 2007. Her Ph.D. thesis work, under the guidance of Professor Seth M. Cohen, consisted of examining novel zinc-specific chelators for incorporation into inhibitors of matrix metalloproteinases. In 2008 she started her NIH-funded postdoctoral research with Professor David P. Giedroc, studying zinc homeostasis in Streptococcus pneumoniae. David Giedroc graduated from the Pennsylvania State University in 1980 with his B.S. degree in Biochemistry. After a brief stint in Joseph Villafranca's group at Penn State, he earned his Ph.D. degree in Biochemistry at Vanderbilt University in 1984, where he worked with David Puett on the calcium sensor calmodulin. From 1984 to 1988, he was an NIH postdoctoral fellow in the late Joseph E. Coleman's laboratory at Yale University, where he worked on zinc-finger DNA binding proteins. From 1988 to 2007, he was a member of the faculty in the Department of Biochemistry and Biophysics at Texas A&M University. He is now Professor of Chemistry at Indiana University, where he continues his studies of metalloregulatory proteins and viral RNA structure, folding, and function.

The SmtB/ArsR family of prokaryotic metalloregulatory transcriptional repressors represses the expression of operons linked to stressinducing concentrations of di-and multivalent heavy metal ions. Derepression results from direct binding of metal ions by these homodimeric 'metal sensor' proteins. An evolutionary analysis, coupled with comparative structural and spectroscopic studies of six SmtB/ArsR family members, suggests a unifying 'theme and variations' model, in which individual members have evolved distinct metal selectivity profiles by alteration of one or both of two structurally distinct metal coordination sites. These two metal sites are designated K3N (or K3) and K5 (or K5C), named for the location of the metal binding ligands within the known or predicted secondary structure of individual family members. The K3N/K3 sensors, represented by Staphylococcus aureus pI258 CadC, Listeria monocytogenes CadC and Escherichia coli ArsR, form cysteine thiolate-rich coordination complexes (S 3 or S 4 ) with thiophilic heavy metal pollutants including Cd(II), Pb(II), Bi(III) and As(III) via inter-subunit coordination by ligands derived from the K3 helix and the N-terminal 'arm' (CadCs) or from the K3 helix only (ArsRs). The K5/K5C sensors Synechococcus SmtB, Synechocystis ZiaR, S. aureus CzrA, and Mycobacterium tuberculosis NmtR form metal complexes with biologically required metal ions Zn(II), Co(II) and Ni(II) characterized by four or more coordination bonds to a mixture of histidine and carboxylate ligands derived from the C-terminal K5 helices on opposite subunits. Direct binding of metal ions to either the K3N or K5 sites leads to strong, negative allosteric regulation of repressor operator/promoter binding affinity, consistent with a simple model for derepression. We hypothesize that distinct allosteric pathways for metal sensing have coevolved with metal specificities of distinct K3N and K5 coordination complexes.

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Programmed ribosomal frameshifting (PRF) is one of multiple translational recoding processes that fundamentally alters triplet decoding of the messenger RNA by the elongating ribosome. The ability of the ribosome to change translational reading frames in the −1 direction (−1 PRF) is employed by many positive strand RNA viruses, including economically important plant viruses and many human pathogens such as retroviruses, e.g., HIV-1, and coronaviruses, e.g., the causative agent of severe acute respiratory syndrome (SARS), in order to properly express their genomes. −1 PRF is programmed by a bipartite signal embedded in the mRNA and includes a heptanucleotide "slip site" over which the paused ribosome "backs up" by one nucleotide, and a downstream stimulatory element, either an RNA pseudoknot or a very stable RNA stem-loop. These two elements are separated by 6-8 nucleotides, a distance that places the 5′ edge of the downstream stimulatory element in direct contact with the mRNA entry channel of the 30S ribosomal subunit. The precise mechanism by which the downstream RNA stimulates −1 PRF by the translocating ribosome remains unclear. This review summarizes the recent structural and biophysical studies of RNA pseudoknots and places this work in the context of our evolving mechanistic understanding of translation elongation. Support for the hypothesis that the downstream stimulatory element provides a kinetic barrier to the ribosome mediated unfolding is discussed.

Staphylococcus aureusA bout one-third of all proteins exploit specific metal ions to assist in macromolecular folding and͞or function at the active site of metalloenzymes (1). All cells restrict the number of bioavailable metal atoms to avoid any excess that would otherwise compete with native metal ion sites that do not support biological activity (2). Essentially all cell types contain intracellular metal sensors that detect surplus metal ions and control the expression of genes encoding proteins that expel or sequester the extra ions (3). For some metals and some cell types, a complementary set of sensors detect deficiency and regulate genes encoding proteins that acquire more of the required ions (4, 5). It is currently poorly understood how such metal-sensing metalloregulators accurately discriminate between various metal ions.SmtB͞ArsR-family regulators are ubiquitous in bacterial genomes and bind to the operator͞promoter (O͞P) regions of gene(s) encoding proteins involved in metal export or sequestration, repressing transcription (for a review, see ref. 6). As the concentration of metal ion increases, the effector-binding sites of the regulators become occupied eliciting a conformational change that weakens the affinity for the O͞P region, allowing transcription to proceed. Members of the SmtB͞ArsR family include: As(III), Sb(III), Bi(III)-responsive ArsR (7), Zn(II)-responsive SmtB (8), Cd(II), Pb(II), Bi(III)-responsive CadC (9-11), Zn(II)-responsive ZiaR (12), Co(II), Zn(II)-responsive CzrA (13,14), and, most recently, Ni(II), Co(II)-responsive NmtR (15).Comparative structural and spectroscopic studies of six SmtB͞ ArsR family members reveal that individual members are characterized by one or both of two structurally distinct metal coordination sites (6, 11, 15-20). These two metal sites are designated ␣3N (or ␣3) and ␣5 (or ␣5C), named for the location of the metal-binding ligands within the known or predicted secondary structure of individual family members. The coordination environment and precise ligand set of the ␣3, ␣3N, and͞or ␣5, ␣5C sites in the different SmtB͞ArsR proteins differ and are presumed to contribute toward metal selectivity. A sequence comparison for proteins discussed herein is shown in Fig. 1 and highlights these sites.Here we report insights gained from the study of two additional family members, Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR. CzrA and NmtR share 30% sequence identity and a high degree of similarity (60%) yet respond to distinct but partially overlapping metal profiles in vivo. S. aureus CzrA is a Co(II)͞Zn(II)-specific sensor that regulates the expression of the czr operon, which encodes a Co(II)͞ Zn(II)-facilitated pump, CzrB, that effluxes metal out of the cell (13, 14). Electromobility-shift assays and in vivo expression studies indicate that Zn(II) is the strongest inducer of CzrA regulation, with Co(II) also capable of regulation but only at higher concentrations than Zn(II). Other metals, including Ni(II), have little to no effect on derep...

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