分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: Rv3899c, a hypothetical protein from Mycobacterium tuberculosis that is conserved within the mycobacteria, is predicted to be secreted and has been found in culture filtrates. Here, Rv3899c has been cloned, expressed in Escherichia coli and purified using standard chromatographic techniques. The hanging-drop vapour-diffusion method with PEG 3350 as a precipitant was used to crystallize the protein. N-terminal sequencing results showed that the amino-acid sequence of the crystallized protein began with GATAG, indicating that it is a fragment containing residues 184-410 of Rv3899c. Rv3899c(184-410) crystals exhibited the symmetry of space group P2(1)2(1)2(1), with unit-cell parameters a = 49.88, b = 54.72, c = 75.52 angstrom, = = = 90 degrees, and diffracted to a resolution of 1.90 angstrom.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-11
摘要: C/EBP alpha is a critical transcriptional regulator of adipogenesis. HowC/EBP alpha transcriptionis itself regulated is poorly understood, however, and remains a key question that needs to be addressed for a complete understanding of adipogenic development. Here, we identify a lncRNA, ADINR(adipogenic differentiation induced noncoding RNA), transcribed from a position similar to 450 bp upstream of the C/EBP alpha gene, that orchestrates C/EBP alpha transcriptionin vivo. Depletion of ADINR leads to a severe adipogenic defect that is rescued by overexpression of C/EBP alpha. Moreover, we reveal that ADINR RNA specifically binds to PA1 and recruits MLL3/4 histone methyl-transferase complexes so as to increase H3K4me3 and decrease H3K27me3 histone modification in the C/EBP alpha locus during adipogenesis. These results show that ADINR plays important roles in regulating the differentiation of human mesenchymal stem cells into adipocytes by modulating C/EBP alpha in cis.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-05
摘要: DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an alpha beta(2)epsilon polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its a subunit, Mtb DNA pol III has two potential proofreading subunits; the alpha and epsilon subunits. During DNA replication, the presence of the beta(2) clamp strongly promotes the polymerization of the alpha beta(2)epsilon replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb.