1. Volume 19, Issue 9, Pages 1241-1251 (September 2011)
Unusual, Dual Endo- and Exonuclease Activity in the Degradosome Explained by Crystal Structure Analysis of RNase J1 
Joseph A. Newman, Lorraine Hewitt, Cecilia Rodrigues, Alexandra Solovyova, Colin R. Harwood, Richard J. Lewis 
Structure 
Volume 19, Issue 9, Pages (September 2011)
DOI: /j.str
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2. Figure 1 The B. subtilis RNase J1 Structure
(A) Cartoon representation of the monomer structure of RNase J1, color ramped from blue at the N terminus to red at the C terminus, the secondary structural elements are labeled as they appear in the text.
(B) Analysis of the electron density in the vicinity of the active site. 2Fobs-1Fcalc electron density is contoured at 1.5 σ and colored blue; anomalous difference density, calculated using model phases, is contoured at 4.5 σ and colored orange. The two zinc ions are shown as pink spheres.
Space-filling representation of the RNase J1 dimer (C) and tetramer (D), with each protomer in the dimer and each homodimer in the tetramer colored independently.
Structure  , DOI: ( /j.str )
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3. Figure 2 The Oligomeric State of RNase J1 in Solution
(A) Size exclusion chromatography of RNase J1 on an analytical S200 (10/30) gel filtration column yields peaks at 11.9 and 13.1 ml, corresponding to tetramers and dimers (250 and 110 kDa, respectively). The weights have been calculated from the calibration curve shown in the inset.
(B) Sedimentation velocity AUC of dimeric (above) and tetrameric forms (below) of RNase J1 showing sedimentation coefficients of 5.85 S and 9.74 S, in excellent agreement with those calculated from the coordinates of the RNase J1 dimer (6.19 S) and tetramer (9.69 S), which are shown on the figure as solid black lines.
Structure  , DOI: ( /j.str )
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4. Figure 3 RNA Binding to RNase J1
(A) Comparison of the open (B. subtilis RNase J1, opaque) and closed (T. thermophilus RNase J, semitransparent) forms of RNase J-type monomers after superimposition on the β-lactamase domain. See also Figure S1.
(B) Overview of the RNase J1 RNA-binding site with a five residue RNA substrate modeled into its expected position. Residues contributing significant contacts to the RNA are labeled.
(C) Detailed view of the RNase J1 active site with key residues labeled, zinc ions shown as pink spheres, and the catalytic water as a red sphere. Black arrows show the proposed route of nucleophilic attack on the phosphate and electron transfer to the UMP leaving group.
(D) View of the RNase J2 active site from the same orientation as that in (C). The nonconservation of three of the metal ion-coordinating residues, including the catalytic Asp78, renders RNase J2 unlikely to coordinate two zinc ions.
See also Figure S2.
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5. Figure 4 The RNase J1/RNase J2 Interaction
(A) SPR sensorgram showing serial injections of the RNase J2 dimer (concentrations ranging from 100 to 500 nM) over a sensor surface containing ∼1600 RU of the RNase J1 dimer. The data were fitted to a 1:1 Langmuir binding model with a KD of 0.08 μM.
(B) Titration electrophoretic mobility shift assay on nondenaturing PAGE. A constant amount of RNase J1 was incubated with increasing amounts of RNase J2 (the numbers above the gel indicate quantities in micrograms).
Structure  , DOI: ( /j.str )
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6. Figure 5
The RNase J1 Endonuclease Activity and Allosteric Conformational Changes
(A) Cross section of the RNase J1 dimer with a single RNA substrate modeled into the active site and RNA entry and exit tunnels labeled.
(B) Cross section of the RNase J1 tetramer with RNA modeled into the active site, viewed from the same view as in (A). The dotted line represents the shortest two possible exit paths for long endonuclease substrates to exit the tetramer, both of which require significant bending of the RNA.
(C) The calcium ion-binding site identified in the RNase J1 structure with metal ion-coordinating residues labeled. Each protomer in the dimer is colored independently This calcium-binding site is close to the linker helix α13 and the hinge region, both of which undergo substantial conformational changes in the transition from the open to closed forms of the enzyme. The black arrows indicate the direction of movement required to form a fully occupied octahedral coordination.
See also Movie S1.
Structure  , DOI: ( /j.str )
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