Characterization of the Partitioning System of Myxococcus Plasmid pMF1

pMF1 is the only autonomously replicating plasmid that has been recently identified in myxobacteria. This study characterized the partitioning (par) system of this plasmid. The fragment that significantly increased the retaining stability of plasmids in Myxococcus cells in the absence of selective antibiotics contained three open reading frames (ORFs) pMF1.21-pMF1.23 (parCAB). The pMF1.22 ORF (parA) is homologous to members of the parA ATPase family, with the highest similarity (56%) to the Sphingobium japonicum ParA-like protein, while the other two ORFs had no homologs in GenBank. DNase I footprinting and electrophoretic mobility shift assays showed that the pMF1.23 (parB) product is a DNA-binding protein of iteron DNA sequences, while the product of pMF1.21 (parC) has no binding activity but is able to enhance the DNA-binding activity of ParB to iterons. The ParB protein autogenously repressed the expression of the par genes, consistent with the type Ib par pattern, while the ParC protein has less repressive activity. The ParB-binding iteron sequences are distributed not only near the partitioning gene loci but also along pMF1. These results indicate that the pMF1 par system has novel structural and functional characteristics

Uncovering Enhancer Functions Using the α-Globin Locus

Over the last three decades, studies of the α- and β-globin genes clusters have led to elucidation of the general principles of mammalian gene regulation, such as RNA stability, termination of transcription, and, more importantly, the identification of remote regulatory elements. More recently, detailed studies of α-globin regulation, using both mouse and human loci, allowed the dissection of the sequential order in which transcription factors are recruited to the locus during lineage specification. These studies demonstrated the importance of the remote regulatory elements in the recruitment of RNA polymerase II (PolII) together with their role in the generation of intrachromosomal loops within the locus and the removal of polycomb complexes during differentiation. The multiple roles attributed to remote regulatory elements that have emerged from these studies will be discussed

Structural basis for ADP-mediated transcriptional regulation by P1 and P7 ParA

The accurate segregation of DNA is essential for the faithful inheritance of genetic information. Segregation of the prototypical P1 plasmid par system requires two proteins, ParA and ParB, and a centromere. When bound to ATP, ParA mediates segregation by interacting with centromere-bound ParB, but when bound to ADP, ParA fulfils a different function: DNA-binding transcription autoregulation. The structure of ParA is unknown as is how distinct nucleotides arbitrate its different functions. To address these questions, we carried out structural and biochemical studies. Crystal structures show that ParA consists of an elongated N-terminal α-helix, which unexpectedly mediates dimerization, a winged-HTH and a Walker-box containing C-domain. Biochemical data confirm that apoParA forms dimers at physiological concentrations. Comparisons of four apoParA structures reveal a strikingly flexible dimer interface that allows ParA to adopt multiple conformations. The ParA–ADP structure shows that ADP-binding activates DNA binding using a bipartite mechanism. First, it locks in one specific dimer conformation, and second, it induces the folding of two DNA-binding basic motifs that we show are critical for operator binding