Kinetic parameters for the wild-type xoFabV enzyme.

<p>Kinetic parameters for the wild-type xoFabV enzyme.</p

Structural comparison of the Y-X₈-K motif of xoFabV and the Y-X₆-K motif of ecFabI.

<p>Despite the fact that the Y-X₈-K motif of xoFabV has two more residues than the Y-X₆-K motif of ecFabI, the conformations of the conserved tyrosine and lysine residues are similar. The distance between the conserved tyrosine (Y236) and lysine (K245) residues in the Y-X₈-K motif of xoFabV (shown in green) is 10.4 Å, while the distance between Y156 and K163 in the Y-X₆-K motif of ecFabI (magenta) is 4.5 Å.</p

Full-length sequence alignment of three enoyl-ACP reductase enzymes from different organisms.

<p>The sequences are from <i>V. cholerae, B. mallei</i> and <i>X. oryzae</i>. Y226, Y236 and K245 of xoFabV and their corresponding residues in the other two enzymes are labelled with asterisks. V246 of xoFabV and its corresponding residues are labelled with a colon. The sequence alignment was performed using T-Coffee, and the figure was made using ESPript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026743#pone.0026743-Gouet1" target="_blank">[34]</a>.</p

Primers used for cloning and mutagenesis.

a<p>Restriction sites and mutated sites are underlined.</p

X-ray diffraction data collection and refinement statistics.

a<p>Values in parentheses are for the highest resolution shell.</p>b<p>ASU, asymmetric unit.</p>c<p><i>R</i>_work = Σ||<i>F</i>_obs|–|<i>F</i>_calc||/Σ|<i>F</i>_obs|, where <i>F</i>_calc and <i>F</i>_obs are the calculated and observed structure factor amplitudes, respectively.</p>d<p><i>R</i>_free = as for <i>R</i>_work, but for 3.7% of the total reflections chosen at random and omitted from refinement.</p

Crystal structure of xoFabV.

<p>The structure consists of 13 α-helices and 11 β-strands, representing a classic Rossmann fold architecture. The secondary structures are shown in different colours and are labelled with the corresponding numbers. (A) Side view. (B) Top view. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026743#pone-0026743-g001" target="_blank">Figures 1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026743#pone-0026743-g002" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026743#pone-0026743-g003" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026743#pone-0026743-g004" target="_blank">4</a> were made using PyMOL (DeLano Scientific, Palo Alto, California, USA; <a href="http://www.pymol.org" target="_blank">http://www.pymol.org</a>).</p

Structural comparison of individual residues in xoFabV and ecFabI.

<p>The individual residues listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026743#pone-0026743-t002" target="_blank">Table 2</a> are shown in sticks and balls. Their positions in the overall structures are labelled with circles and are enlarged. The residues of xoFabV are shown in green and are listed before the hyphen; the residues of ecFabI are shown in magenta and after the hyphen.</p

Overall structural comparison of xoFabV and ecFabI.

<p>The two structures differ dramatically. The β-sheet and the flanking α-helices are shifted, and xoFabV has additional secondary structures, including helices α1 and α11 – α13 and strands β5 – β8 and β11, which are shown in yellow. Other parts of xoFabV are shown in green; ecFabI is shown in magenta. (A) Side view. (B) Top view.</p

Progress curve analysis of the wild-type and mutant xoFabV variants in the NADH oxidation assay.

<p>The enzyme activity of wild-type and mutant xoFabV was determined by monitoring the oxidation of NADH to NAD⁺ at 340 nm. The reaction was initiated by adding the substrate crotonyl-CoA and was monitored for 10 min at 25°C.</p

Identification of Intermediate-Size Non-Coding RNAs Involved in the UV-Induced DNA Damage Response in <em>C. elegans</em>

<div><h3>Background</h3><p>A network of DNA damage response (DDR) mechanisms functions coordinately to maintain genome integrity and prevent disease. The Nucleotide Excision Repair (NER) pathway is known to function in the response to UV-induced DNA damage. Although numbers of coding genes and miRNAs have been identified and reported to participate in UV-induced DNA damage response (UV-DDR), the precise role of non-coding RNAs (ncRNAs) in UV-DDR remains largely unknown.</p> <h3>Methodology/Principal Findings</h3><p>We used high-throughput RNA-sequencing (RNA-Seq) to discover intermediate-size (70–500 nt) ncRNAs (is-ncRNAs) in C. elegans, using the strains of L4 larvae of wild-type (N2), UV-irradiated (N2/UV100) and NER-deficient mutant (<em>xpa-1</em>), and 450 novel non-coding transcripts were initially identified. A customized microarray assay was then applied to examine the expression profiles of both novel transcripts and known is-ncRNAs, and 57 UV-DDR-related is-ncRNA candidates showed expression variations at different levels between UV irradiated strains and non- irradiated strains. The top ranked is-ncRNA candidates with expression differences were further validated by qRT-PCR analysis, of them, 8 novel is-ncRNAs were significantly up-regulated after UV irradiation. Knockdown of two novel is-ncRNAs, ncRNA317 and ncRNA415, by RNA interference, resulted in higher UV sensitivity and significantly decreased expression of NER-related genes in <em>C. elegans</em>.</p> <h3>Conclusions/Significance</h3><p>The discovery of above two novel is-ncRNAs in this study indicated the functional roles of is-ncRNAs in the regulation of UV-DDR network, and aided our understanding of the significance of ncRNA involvement in the UV-induced DNA damage response.</p> </div