Data Analysis Recitation Activities Support Better Understanding in SEA-PHAGES CURE

Course-based undergraduate research experiences (CUREs) are widely known to improve student learning outcomes in the sciences. Undergraduate students have a particularly difficult time interpreting the scientific data that they generate in these experiences – especially when lacking opportunity and exposure to science processes prior to entering higher education. Therefore, it is vital to structure these research experiences such that students can see maximal gains in their skills. This is especially true in the SEA-PHAGES (Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science) lab experience, which focuses on bacteriophages – a subject about which most undergraduate students have limited knowledge. In the SEA-PHAGES lab experience at The Ohio State University, we observed that while students made rapid gains in science process skills over the course of the semester, they still struggled to interpret the data they generated. To address this issue, we designed and implemented a set of five recitation activities to complement the lab experience, termed Recitation Activities to Improve Literacy in Science (RAILS). Using an adapted student assessment of learning gains (SALG) survey, we observed that these activities improved students' perceived ability to interpret their data, and students reported that they experienced significant gains in their data analysis ability as a result of the activities. We hope that other SEA-PHAGES instructors will similarly benefit from utilizing these recitation activities as part of their implementation of the curriculum. Primary image: Phages on RAILS. A bacteriophage is pictured driving a train engine down a track

DksA2, a zinc‐independent structural analog of the transcription factor DksA

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/116380/1/feb2s0014579313001233-sup-mmc1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/116380/2/feb2s0014579313001233.pd

pH Dependence of the Stress Regulator DksA

<div><p>DksA controls transcription of genes associated with diverse stress responses, such as amino acid and carbon starvation, oxidative stress, and iron starvation. DksA binds within the secondary channel of RNA polymerase, extending its long coiled-coil domain towards the active site. The cellular expression of DksA remains constant due to a negative feedback autoregulation, raising the question of whether DksA activity is directly modulated during stress. Here, we show that <i>Escherichia coli</i> DksA is essential for survival in acidic conditions and that, while its cellular levels do not change significantly, DksA activity and binding to RNA polymerase are increased at lower pH, with a concomitant decrease in its stability. NMR data reveal pH-dependent structural changes centered at the interface of the N and C-terminal regions of DksA. Consistently, we show that a partial deletion of the N-terminal region and substitutions of a histidine 39 residue at the domain interface abolish pH sensitivity in vitro. Together, these data suggest that DksA responds to changes in pH by shifting between alternate conformations, in which competing interactions between the N- and C-terminal regions modify the protein activity.</p></div

Δ<i>dksA</i> mutants are sensitive to acidic conditions.

<p>(A) WT and Δ<i>dksA E</i>. <i>coli</i> strains were grown overnight in rich medium at pH 7.8. Cultures were diluted 1:50 into LB medium at pH 2.5. At selected time points aliquots were taken and the percentage of survival of bacteria was determined using viable count. (B) WT and Δ<i>dksA E</i>. <i>coli</i> strains were grown at pH 7.8, followed by 2.5 hour adaptation at pH 6.5–4.5, and then diluted into LB medium at pH 3.5. Survival was determined using viable counts; the result after 2 hour incubation at pH 3.5 is shown. (C) DksA concentration remains relatively constant at low pH. Samples taken at different time points after a change in pH were analyzed using Western blotting with anti-DksA antibodies. Extract from the Δ<i>dksA</i> strain and purified DksA were loaded as controls.</p

DksA is sensitive to changes in pH.

<p>(A) DksA activity increases at low pH. Increasing concentrations of DksA were added to holo RNAP (30 nM), ApC dinucleotide (0.2 mM), UTP (0.2 mM), GTP (4 μM) and [α-³²P]-GTP (10 μCi of 3000 Ci mmol⁻¹) followed by incubation for 15 minutes in Transcription buffer (20 mM Tris-HCl pH 7.9, 20 mM NaCl, 10 mM MgCl2, 14 mM 2-mercaptoethanol, 0.1 mM EDTA). A linear DNA fragment containing the <i>rrnB</i> P1 promoter was added to initiate transcription and the formation of a 4 nucleotide RNA product was monitored on a denaturing 8% acrylamide gel. A dotted line marks the inhibition of 50% of transcription and is denoted as IC₅₀. The IC₅₀ values (calculated using a single-site binding equation from three independent repeats combined in a best-fit curve, in μM) were: pH 7.6 − 0.7 ± 0.28, pH 6.7 − 0.11 ± 0.016. (B) DksA affinity to core increases at lower pH. DksA binding to core RNAP was performed using the localized Fe²⁺ mediated cleavage assay at different pH. DksA concentrations were: 0, 25, 50, 100, 200 and 400 nM. FL—Full length protein, Cl—cleaved protein, Kd app—apparent Kd.</p

Substitution of His39 alters DksA sensitivity to pH.

<p>(A) The effect of pH on DksA variants. DksA activity and IC₅₀ calculations were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120746#pone.0120746.g003" target="_blank">Fig. 3A</a> with the <i>rrnB</i> P1 promoter. Experiments were performed at least three times at each pH. (B) Thermostability of DksA^H39A is low and relatively insensitive to pH. Thermostability was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120746#pone.0120746.g004" target="_blank">Fig. 4</a>.</p

DksA structure is sensitive to pH.

<p>Two-dimensional ¹H-¹⁵N HSQC spectra at pH 8 (black) and 6 (red) reveal large chemical shift changes at (A) Tyr23 and (B) many other residues.</p

Interaction of the HIV-1 intasome with Transportin 3 (TNPO3 or TRN-SR2)

Transportin 3 (TNPO3 or TRN-SR2) has been shown to be an important cellular factor for early steps of lentiviral replication. However, separate studies have implicated distinct mechanisms for TNPO3, either through its interaction with HIV-1 integrase or capsid. Here we have carried out a detailed biophysical characterization of TNPO3 and investigated its interactions with viral proteins. Biophysical analyses including circular dichroism, analytical ultracentrifugation, small-angle x-ray scattering and homology modeling provide insight into TNPO3 architecture and indicate that it is highly structured and exists in a monomer-dimer equilibrium in solution. In vitro biochemical binding assays argued against meaningful direct interaction between TNPO3 and the CA cores. Instead, TNPO3 effectively bound to the functional intasome, but not to naked viral DNA, suggesting that TNPO3 can directly engage the HIV-1 IN tetramer prebound to the cognate DNA. Mass spectrometry-based protein footprinting and site-directed mutagenesis studies have enabled us to map several interacting amino acids in the HIV-1 IN C-terminal domain and the cargo binding domain of TNPO3. Our findings provide important information for future genetic analysis to better understand the role of TNPO3 and its interacting partners for HIV-1 replication.status: publishe