RESUMO
In the photosynthetic bacterium Rhodobacter sphaeroides, a transcriptional response to the reactive oxygen species singlet oxygen ((1)O(2)) is mediated by ChrR, a zinc metalloprotein that binds to and inhibits the activity of the alternative σ factor σ(E). We provide evidence that (1)O(2) promotes the dissociation of σ(E) from ChrR to activate transcription in vivo. To identify what is required for (1)O(2) to promote the dissociation of σ(E)/ChrR complexes, we analyzed the in vivo properties of variant ChrR proteins with amino acid changes in conserved residues of the C-terminal cupin-like domain (ChrR-CLD). We found that (1)O(2) was unable to promote the detectable dissociation of σ(E)/ChrR complexes when the ChrR-CLD zinc ligands (His141, His143, Glu147, and His177) were substituted with alanine, even though individual substitutions caused a 2-fold to 10-fold decrease in zinc affinity for this domain relative to that for wild-type ChrR (K(d)â¼4.6×10(-)(10) M). We conclude that the side chains of these invariant residues play a crucial role in the response to (1)O(2). Additionally, we found that cells containing variant ChrR proteins with single amino acid substitutions at Cys187 or Cys189 exhibited σ(E) activity similar to those containing wild-type ChrR when exposed to (1)O(2), suggesting that these thiol side chains are not required for (1)O(2) to induce σ(E) activity in vivo. Finally, we found that the same aspects of R. sphaeroides ChrR needed for a response to (1)O(2) are required for the dissociation of σ(E)/ChrR complexes in the presence of the organic hydroperoxide t-butyl hydroperoxide.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica , Rhodobacter sphaeroides/metabolismo , Fator sigma/metabolismo , Oxigênio Singlete/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Coenzimas/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Rhodobacter sphaeroides/genética , Fatores de Transcrição/genética , Zinco/metabolismoRESUMO
Cartilage acidic protein 1 (CRTAC1) gene expression is used as a marker for chondrocyte differentiation in stem cell-based tissue engineering. It is also transcribed outside the skeleton where at least two different transcripts are expressed in lung and brain. In the pituitary gland of the teleost fish sea bream Sparus auratus, we have found a transcript with a high degree of sequence identity to CRTAC1 family members but lacking the EGF-like calcium-binding domain encoding sequence of CRTAC1 and designated it as CRTAC2. Database searches revealed many previously unidentified members of the CRTAC1 and CRTAC2 in phylogenetically distant organisms, such as cyanobacteria, bryophyta, lancelets, and diverse representatives of vertebrates. Phylogenetic analyses showed that the genes encoding CRTAC1 and CRTAC2 proteins coexist in teleost fish genomes. Structural prediction analysis identified the N-terminal region of the CRTAC1/CRTAC2 family members as a potential seven-bladed beta-propeller structure, closely related to those of integrin alpha chains and glycosylphosphatidylinositol-specific phospholipase D1 protein families. This relationship is confirmed by phylogenetic analysis with the N-terminal domain of sea bream CRTAC2 as the most divergent sequence. Because teleost fishes are the only phylogenetic group where both CRTAC1 and CRTAC2 genes are present, they occupy a pivotal position in studies of the mechanisms governing the specific expression patterns of each gene/protein subfamily. This will be essential to elucidate their respective biological roles.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Peixes/genética , Filogenia , Hipófise/metabolismo , Dourada/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Sequência Conservada , Cianobactérias/genética , Evolução Molecular , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Hipófise/química , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
A transcriptional response to singlet oxygen in Rhodobacter sphaeroides is controlled by the group IV sigma factor sigma(E) and its cognate anti-sigma ChrR. Crystal structures of the sigma(E)/ChrR complex reveal a modular, two-domain architecture for ChrR. The ChrR N-terminal anti-sigma domain (ASD) binds a Zn(2+) ion, contacts sigma(E), and is sufficient to inhibit sigma(E)-dependent transcription. The ChrR C-terminal domain adopts a cupin fold, can coordinate an additional Zn(2+), and is required for the transcriptional response to singlet oxygen. Structure-based sequence analyses predict that the ASD defines a common structural fold among predicted group IV anti-sigmas. These ASDs are fused to diverse C-terminal domains that are likely involved in responding to specific environmental signals that control the activity of their cognate sigma factor.