1. Volume 22, Issue 5, Pages 719-730 (May 2014)
Structure and Function of RNase AS, a Polyadenylate-Specific Exoribonuclease Affecting Mycobacterial Virulence In Vivo 
Maria Romano, Robert van de Weerd, Femke C.C. Brouwer, Giovanni N. Roviello, Ruben Lacroix, Marion Sparrius, Gunny van den Brink-van Stempvoort, Janneke J. Maaskant, Astrid M. van der Sar, Ben J. Appelmelk, Jeroen J. Geurtsen, Rita Berisio 
Structure 
Volume 22, Issue 5, Pages (May 2014)
DOI: /j.str
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2. Figure 1
Effect of Inactivation of MMAR_3223 on Virulence of M. marinum in the Zebrafish Embryo
Mcherry expressing M. marinum (Mma20) WT or MMAR_3223::Tn mutant live bacteria were injected into zebrafish embryos and the bacterial infection monitored by fluorescence or plating. Inactivation of MMAR_3223 leads to attenuation in virulence.
(A and B) Bright-field (left) and fluorescent (right) pictures of 5 days postinfection (dpi) embryos infected with M. marinum WT (A) or (B) mutant MMAR_3223::Tn bacteria. The bacterial injection inocula were 161 and 169 CFU, respectively.
(C) Quantification of infection with M. marinum WT or MMAR_3223::Tn in embryos at 5 dpi. The fluorescence intensities were quantified by proprietary imaging software and SEM are shown.
(D) Infection of M. marinum WT, MMAR_3223::Tn mutant and MMAR_3223::Tn complemented with M. tuberculosis rv2179c, measured by CFU plating of 5 dpi old zebrafish embryos. Each data point represents the CFU count of one infected embryo. The CFU counts are graphically displayed in a log scale. Means are represented with bars and mean values are shown in numbers. The embryos were inoculated with 63, 47, and 62 CFU for WT, mutant, and complementant, respectively; ∗∗p < , unpaired Student’s t test. Clearly, inactivation of M. marinum MMAR_3223 leads to a strong attenuation in vivo and this can be complemented with M. tuberculosis rv2179c (the gene product of which is RNase AS).
See also Figures S1 and S2 and Tables S1 and S2.
Structure  , DOI: ( /j.str )
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3. Figure 2 RNase AS Is a Dimer in the Crystal State
(A) Cartoon representation of RNase AS crystal structure. Chains A and B of the dimer are shown in orange and green, respectively. The inset shows an enlargement of one of the two catalytic sites, with catalytic DEDDh residues shown in stick representation.
(B) Degree of residue conservation on the protein surface (Consurf analysis) plotted on chain B, whereas chain A is shown as blue ribbon. Residue coloring, reflecting the degree of residue conservation, ranges from magenta (highly conserved) to cyan (variable). Conserved residues in the catalytic site and in the interface region are labeled.
See also Figure S3.
Structure  , DOI: ( /j.str )
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4. Figure 3 RNase AS Selectively Hydrolyses Poly(A)
(A) HPLC profiles (280 nm) of poly(A) after 48 hr incubation with RNase AS (dashed black), with the RNase AS mutant H140A (black) and in protein buffer (gray). Electrospray ionization mass (inset) clearly identifies AMP (m/z ) as a reaction product.
(B) HPLC profiles (280 nm) of poly(U), poly(G), and poly(C) after 48 hr incubation with RNase AS (upper curve) and protein buffer (bottom curve). An arbitrary shift was applied for clarity.
(C) HPLC profiles (280 nm) after 48 hr incubation with RNase AS with the polynucleotide AAAACAAAA (black) and in protein buffer (gray). Degradation products are labeled. The reported value of m/z (1,559.49, negative ion) is in agreement with the expected value for the product AAAAC ([C49H61N23O29P4-H]−, m/z 1,559.05).
Structure  , DOI: ( /j.str )
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5. Figure 4 RNase AS Strongly Discriminates between RNA and DNA
(A) HPLC profile (280 nm) of A12 after 3 hr incubation with RNase AS (upper curve) and with the protein buffer (bottom curve).
(B) HPLC profiles after incubation of dA12 with RNase AS, in the time range 3–72 hr. In both panels, UV profiles after incubations were arbitrarily upshifted, for clarity.
Structure  , DOI: ( /j.str )
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6. Figure 5 Binding Mode of AMP Nucleotides
(A) Omit (Fo-Fc) electron density map, contoured at 2.0 σ, of AMP(−1) and AMP(+1) nucleotides.
(B) Stick representation of AMP(−1) and AMP(+1) in the catalytic site cleft of RNase AS. Cartoon and surface representations of A and B chains of RNase AS are shown in orange and green, respectively.
(C) Hydrogen bonding interactions and coordination with Mg2+ sites (MGA and MGB, in yellow) of AMP(−1) and AMP(+1) with the enzyme.
See also Figure S4.
Structure  , DOI: ( /j.str )
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7. Figure 6 RNase AS Does Not Degrade Polyinosine
HPLC profiles (280 nm) of polyinosine after 48 hr incubation with RNase AS (black) and in protein buffer (blank, gray). An arbitrary shift was applied for clarity. The inset shows the chemical structure of inosine mononucleotide.
Structure  , DOI: ( /j.str )
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8. Figure 7 Binding Mode of UMP
(A) Omit (Fo-Fc) electron density map, contoured at 2.0 σ, of UMP nucleotide at the (−1) site of RNase AS.
(B) Superposition of complexes of RNase AS with AMP (navy) and UMP (cyan). Whereas UMP does not bind the (+1) site, it shares the same conformation and hydrogen bonding interactions with RNase AS in the (−1) site. Cartoons are shown in light green (UMP complex) and forest green (AMP complex). Binding of either UMP or AMP at the (−1) site induces slightly different conformations of the side chains of Trp46, His140, and Glu8, which are shown in stick representation.
Structure  , DOI: ( /j.str )
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9. Figure 8 Structural Comparisons between RNase AS and RNase T
(A) Superposition of RNase AS structure (orange) to that of RNase T (magenta). The inset shows a detail of structural differences of the region involving the AMP(+1) binding pocket.
(B,C) Electrostatic potential surfaces of RNase AS and RNase T, respectively. Positive potential is drawn in blue, negative in red.
See also Figures S5 and S6.
Structure  , DOI: ( /j.str )
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