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1.
Figure 3

Figure 3. Effects of ADPR, pADPR and NaADP on the thermal stability of soNrtR. From: Structure and function of an ADP-ribose dependent transcriptional regulator of NAD metabolism.

The increase in the thermal stability (ΔTm) of NrtR was monitored in the presence of varying concentrations of ADPR () pADPR () and NaADP ().

Nian Huang, et al. Structure. ;17(7):939-951.
2.
Figure 4

Figure 4. ADPR content in Shewanella oneidensis cells. From: Structure and function of an ADP-ribose dependent transcriptional regulator of NAD metabolism.

HPLC chromatograms of unspiked S. oneidensis extract (black line) and ADPR spiked extract (blue line). Inset shows part of the chromatogram of the unspiked extract (black line) in comparison with that of the same extract after incubation with nucleotide pyrophosphatase (red line)

Nian Huang, et al. Structure. ;17(7):939-951.
3.
Figure 6

Figure 6. Effector binding site of soNrtR. From: Structure and function of an ADP-ribose dependent transcriptional regulator of NAD metabolism.

The bound ADPR (magenta) and interacting protein side chains are shown as sticks. Hydrogen bonds are indicated by dashed lines. Residues from the second monomer are colored in pale green and Nudix Motif region is colored blue.

Nian Huang, et al. Structure. ;17(7):939-951.
4.
Figure 8

Figure 8. ADP-ribose induced conformational changes in soNrtR. From: Structure and function of an ADP-ribose dependent transcriptional regulator of NAD metabolism.

(A). Superposition of soNrtR N-terminal domain dimer in complex with ADPR (colored in wheat) to soNrtR-DNA complex (protein in pale cyan, DNA in orange). The Nudix switch region is indicated by an arrow and highlighted in red and dark blue colors. The region where steric clash would occur is marked by a red asterisk.
(B) and (C). Electron densities of the Nudix Switch region (shown as sticks) in the apo soNrtR (B) and ADPR-bound (C) structures. The densities are contoured at 1.0σ.

Nian Huang, et al. Structure. ;17(7):939-951.
5.
Figure 5

Figure 5. Structures of soNrtR, its complexes with DNA, and with effector ADPR. From: Structure and function of an ADP-ribose dependent transcriptional regulator of NAD metabolism.

(A). Ribbon presentation of soNrtR monomer with the Nudix domain colored in green and wHTH domain in cyan. The secondary structure elements (strands β1-β9 and helices α1-α7) are labeled. The disordered N-terminal region is indicated by dots.
(B). Structure of soNrtR N-terminal Nudix domain dimer in complex with ADP-ribose. ADP-ribose molecules are shown as magenta sticks.
(C). Superposition of soNrtR wHTH domain (blue) with histone H5 (pink, pdb code 1hst), penicillinase repressor (light cyan, 1sd4), and ferric uptake regulator (yellow, 1mzb).
(D). Structure of full-length soNrtR dimer in complex with DNA. The protein is presented as ribbons and DNA as sticks.

Nian Huang, et al. Structure. ;17(7):939-951.
6.
Figure 9

Figure 9. Nudix switches in various Nudix proteins. From: Structure and function of an ADP-ribose dependent transcriptional regulator of NAD metabolism.

Ligand binding induced conformational changes in following Nudix proteins are shown: soNrtR, C. elegans Ap4A hydrolase (ceAp4Aase), the ADPRase domain of Francisella tularensis NadM_Nudix protein (ftNadM_Nudix), M. tuberculosis ADPRase (mtADPRase), and E. coli ADPRase (ecADPRase). The unliganded (cyan) and liganded (magenta) conformations for each protein are superimposed. The ligands are shown as sticks and metal ions as green balls. The disordered regions in some of the structures are shown as dotted lines. The corresponding Nudix switch region in each structure is indicated by a red arrow.

Nian Huang, et al. Structure. ;17(7):939-951.
7.
Figure 1

Figure 1. Transcriptional analysis of the nrtR-prs-nadV region. From: Structure and function of an ADP-ribose dependent transcriptional regulator of NAD metabolism.

(A) Schematic representation of nrtR, prs and nadV genes on the Shewanella oneidensis chromosome and the promoter region of the prs-nadV operon. I and II represent the PCR-amplified fragments. In the promoter region sequence the two predicted NrtR binding sites are in bold, the putative -10 and -35 regulating sequences are underlined, the start codon of prs is indicated by an arrow and shown in boldface.
(B) Ethidium bromide stained agarose gel showing the PCR products obtained with cDNA (lanes a), RNA (lanes b), genomic DNA (lanes c) and water (lanes d) as the templates and the primer pairs amplifying fragments I and II. Lanes M, 1-kb DNA ladder.

Nian Huang, et al. Structure. ;17(7):939-951.
8.

Figure 7. Interactions between soNrtR and NrtR-box DNA. From: Structure and function of an ADP-ribose dependent transcriptional regulator of NAD metabolism.

(A). Interactions between soNrtR (green) and one half site of soNrtR-box DNA. Hydrogen bonds are indicated by dotted lines.
(B) Schematic illustration of soNrtR-DNA interactions. The ATA-n2-GT consensus of NrtR-box half site is boxed in red. Solid arrows indicate hydrogen bonds; dashed arrows van der Waals contacts. Primed deoxyribose denotes the complementary DNA strand. The dyad axis of DNA is indicated by a filled oval at the center of base pair Ade15′-Thy15.
(C). Molecular surface rendering of soNrtR dimer (green and cyan) and DNA duplex (red) in soNrtR-DNA complex.
(D). Heatmap of correlations between amino acid-nucleotide pairs for NrtR proteins and their operators. Sequence logos of NrtR DNA-binding domains and NrtR-box DNA are display above and to the right of the heatmap, respectively. The total height of the symbols in each position equals the positional information content, whereas the height of individual symbols is proportional to the positional amino acid/nucleotide frequency. The correlation scores are color ramped from yellow to red for amino acid-nucleotide pairs with statistical significance Z-score > 5.

Nian Huang, et al. Structure. ;17(7):939-951.
9.

Figure 2. DNase I footprinting and in vitro transcription assay. From: Structure and function of an ADP-ribose dependent transcriptional regulator of NAD metabolism.

(A) DNase I footprinting of soNrtR on prs-nadV operator DNA. Lanes marked A, T, C are from a DNA sequencing ladder of the coding strand of the 144-bp prs-nadV operator DNA. The remaining lanes show the results of DNase I footprinting. Lane 1 contains no soNrtR; lane 2 and 3 contain increasing amount of soNrtR. The DNA sequence around the second protected region is shown to the right of the gel. Bases protected against DNase I treatment are colored red. The 21-bp NrtR-box sequence is boxed. (B) prs-nadV transcript levels determined by real-time PCR in the presence of various amounts of NrtR. (C) prs-nadV transcript levels in the presence of 0.2 μM NrtrR and increasing amounts of ADPR. Levels of transcripts were normalized to the level in the absence of soNrtR (B) and ADPR (C). For the relative quantification 2 −ΔCt was calculated for each sample (ΔCt being the difference between the Ct values of the target and the normalizer). Data represents the averaged results from three independent experiments with duplicates; vertical bars represent standard deviations.

Nian Huang, et al. Structure. ;17(7):939-951.

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