The Vpr (viral protein R) of human immunodeficiency virus, type 1, which is expressed during the late stage of the viral infection, has received special attention because of its ability to control transcription of the human immunodeficiency virus, type 1, long terminal repeat and to influence cell cycle progression. Here we demonstrate that Vpr has the ability to regulate transcription of the cyclin-dependent kinase inhibitor, p21 WAF1 (p21), one of the key regulators of the cell cycle, in human astrocytic cells. The results from transcription assays demonstrated that Vpr augments promoter activity of p21 through the GC-rich region located between nucleotides ؊84 and ؊74 with respect to the ؉1 transcription start site. Activation of p21 by Vpr required cooperativity of Sp1, which binds to the DNA sequence spanning ؊84 to ؊74. Results from bandshift assay revealed an increased level of Sp1 DNA binding activity in the presence of Vpr. Furthermore, Vpr was able to associate with Sp1 via the zinc finger domain located in the Cterminal region of Sp1. Functional studies revealed that the cooperativity between Vpr and Sp1 requires the zinc finger domain at the C terminus and the glutamine-rich domain at the N terminus of Sp1. Expression of p53 further enhanced the level of Vpr-Sp1-mediated transcription activation of p21 through the sequence spanning ؊84 to ؊74 and increased the DNA binding activity of Sp1 in the presence of Vpr. Results from glutathione S-transferase pull-down assay showed the association of Vpr with p53 in extracts containing Sp1. Altogether, the outcome of our functional and binding studies suggested that the physical interaction of Vpr with Sp1 and p53 could modulate transcriptional activity of p21.The cyclin-dependent kinase inhibitor, p21 WAF1 (p21), arrests cell cycle by modulating the activity of cyclin-dependent kinases and regulates DNA methylation by interacting directly with proliferating cell nuclear antigen, a subunit of DNA polymerase, and prevents DNA synthesis (1-4). p21 also plays important roles in the control of cell senescence, apoptosis, and differentiation (5-7). Expression of p21 is regulated by a wide range of proteins such as tumor suppressors including p53 and pRb (8, 9), growth factors, and several signaling proteins associated with cytokines including platelet-derived growth factor

It is shown that Methyl Red can be used as an indicator dye that changes color in E. coli culture as a result of time-and cell density-dependent bleaching by azoreductase produced by the bacteria. For cell cultures that are being used to express a recombinant protein this phenomenon can be exploited to provide a simple visual cue that cell cultures have reached an appropriate growth phase for addition of an agent to induce protein expression, such as isopropylthiogalactoside.Since the advent of the modern era of preparative molecular biology in the 1980s cultures of E. coli have been widely used to overexpress recombinant proteins. The most commonly used methods involve the growth of liquid cultures to mid-logarithmic phase followed by addition of a gene transcription-inducing agent, typically IPTG. The most commonly used method for determining when mid-log phase has been reached is to monitor light scattering of cultures by determining the apparent absorption of light at 600 nm. This can be a timeconsuming and odious task, requiring repeated measurements for each culture flask until an appropriate OD 600 is reached (usually when OD 600 is between 0.5 and 1.0). To see if a visual colorimetric method could be devised to determine when mid-log phase is reached, E. coli cultures were grown of in the presence of a variety of dyes/indicators ( Table S1) at concentrations that confer a visually obvious color to the medium. For these tests we used BL21(DE3) E. coli harboring a pET21b vector encoding the 99 residue C-terminal domain of the amyloid precursor protein (C99(1)). When pH 7.0-buffered M9 cultures were then incubated with rotary shaking at 37°C no change in culture color was observed for most dyes. However, in the case of 100 ml and 1 L cultures grown in the presence of 20 mg/L Methyl Red, it was observed that when the OD 600 reached 0.67 ± 0.1 or 0.75 ± 0.1, respectively, the cultures completed a change in color from orange to nearly a pale yellow ( Figure 1 and Supporting Figure S1). Similar results were obtained for BL21(DE3) cells harboring an empty pET21b plasmid. Tests of 20 mg/ml Methyl Red in 1 L cultures of a very different strain of E. coli (WH1061) harboring a different recombinant plasmid (pSD0005 encoding diacylglycerol kinase(2)) completed the same orange-to-colorless change at OD 600 = 0.87± 0.25, although some calibration of culture conditions was required to assure that the color change occurs when OD 600 reaches the 0.5-1.0 range (see Supporting Information). For each strain/vector it is especially to optimize the volume of the starter culture used to inoculate the fresh dye-containing M9 medium. † This work was supported by RO1 GM47485, PO1 GM080513, and RO1 NS058815. Table S1 and Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 July 6. In either case expression was allowed to proceed for several hours followed by harvesting of the cells and purifica...

Steroid receptor coactivators (SRCs) comprise a family of three paralogous proteins commonly recruited by eukaryotic transcription factors. Each SRC harbors two tandem Per-ARNT-Sim (PAS) domains that are broadly distributed that bind small molecules and regulate interactions. Using computational docking, solution NMR, mass spectrometry, and molecular dynamics simulations, we show that the SRC1 PAS-B domain can bind to certain prostaglandins (PGs) either non-covalently to a surface that overlaps with the site used to engage transcription factors or covalently to a single, specific, conserved cysteine residue next to a solvent accessible hydrophobic pocket. This pocket is in proximity to the canonical transcription factor binding site, but on the opposite side of the domain, suggesting a potential mode of regulating transcriptional activator-coactivator interactions.