The organization of protein and RNA-binding sites in RNase E that define the canonical RNA degradosome, and a description of the derived constructs used in this study. (A) Schematic representation of the RNase E scaffolding domain, which brings together degradosome components. (B) The expression constructs used to prepare recombinant RNase E and RNase E/RhlB complexes. The His₆ ribbon represents a hexa-histidine affinity tag. The jagged lines flanking RNase E(628–843) construct represent non-RNase E peptide sequences derived from its expression vector (a 13-residue leader and a 20-residue tail).

Interaction of RraA with RhlB and its complexes with RNase E. (A) Protein complexes containing RhlB, RNase E peptides, and RraA were analyzed by size-exclusion chromatography. (Left) Overlaid elution profiles from S200 column (the void volume of the column is 45 mL). (Right) SDS-PAGE of selected peak fractions (a–e) showing the composition of the complexes. A 4%–12% gradient PAA gel did not allow for separation of RNase E(696–762) from RraA band (lane 4), but the same samples analyzed subsequently on 4%–20% gradient PAA gel show that both components are present in complex with RhlB (data not shown). (B) Binding of RraA to RhlB was tested in titration electrophoretic mobility shift assays. The same amount of RraA was mixed with increasing amounts of partner protein and analyzed on a polyacrylamide gel under native conditions. RraA was able to change mobility of RhlB (i) or RhlB in complex with its recognition peptide from RNase E, RNase E(696–762) (ii), but no change was observed for RNase E(696–762)/RhlB(1–397), which lacks the C-terminal extension of the helicase (iii).

RraA inhibits 9S RNA processing by RNase E. (A) Secondary structure of 9S RNA, a precursor of 5S rRNA. Indicated are RNase E cleavage sites (a-b, and a minor cleavage site c). Part of the processing product, p5S, was omitted. RNA used in this study has a G instead of an A at the +2 position (underlined). (B) 9S RNA was incubated for the indicated time with RNase E constructs in the presence and absence of RraA. Products of the reactions were analyzed by denaturing PAGE and detected using SYBR Gold. RNase E processes the 9S substrate correctly to the p5S precursor of 5S ribosomal RNA. RraA inhibits the reaction significantly in the case of the degradosome sub-assembly RNase E(1–762)/RhlB (lanes 9,10), but not if only the catalytic domain of RNase E is present (lanes 4,5).

RraA association with RNase E and competition with RNA binding. (A) Increasing amounts of RNase E(628–843) were mixed with the same amount of RraA and analyzed on a polyacrylamide gel under native conditions. Upon addition of RNase E(628–843), a complex with RraA is formed, while the amounts of free RraA decrease. RNase E(628–843) alone does not migrate into the gel due to its positive charge. (B) Recombinant constructs of RNase E CTD were incubated with RraA and analyzed on a native agarose gel. CTD constructs containing at least one of the RNA-binding sites interact with RraA (lanes 2,4,6), but not the construct lacking both the RNA-binding sites: CTDΔRBDΔAR2 (lane 8). The multiple bands are probably due to oligomerization of RNase E CTD, possibly through the RBD region. (C) Analysis of RraA binding to CTD and its deletion constructs from surface plasmon resonance. The kinetic profiles of RraA and CTD, RraA and CTDΔRBD, RraA and CTDΔAR2 interactions fit a 1:1 binding model (see Supplementary material). The affinities were calculated for a RraA trimer. (D) RNase E CTD was first incubated with fluorescein-tagged oligonucleotide (27 mer) or with tRNA, then mixed with RraA and analyzed on a native agarose gel. (Right) UV-fluorescence signal from the 27-mer RNA. CTD binds both tRNA (lane 2) and the 27 mer (lane 6). Upon addition of RraA, the complex of RNase E CTD with tRNA remains unaffected (lane 3). In contrast, in the presence of RraA the 27-mer oligonucleotide is released from RNase E CTD (lane 7) and is replaced by RraA (identical protein band pattern in lanes 5 and 7). B and D are images of the same gel, for clarity shown separately.

Effect of RraA on the ATPase activity of RhlB and ATP-driven remodeling of RNase E/RhlB complexes by RhlB. (A) Effect of RraA on ATPase activity of RhlB in complex with its activator peptide RNase E(696–762). Phosphate release was monitored in the presence and absence of S. cerevisiae bulk RNA, at various RraA to RhlB molar ratios. The ATPase activity of RhlB was inhibited by RraA in the presence of RNA (left), but stimulated in the absence of RNA (see table). Enzyme velocity was calculated from the 100-sec period between the first 50 and 150 sec of the reaction (shaded zone), as the mole of phosphate produced per second per mole of enzyme. The curves, average velocities, and their standard deviations (in parentheses) were calculated from three reactions each, except the RraA controls. (B) The composite image is an overlay of Coomassie protein staining (red) and SYBR Gold staining of nucleic acid (green). RNase E(628–843)/RhlB complex was premixed with tRNA, incubated with RraA, and supplemented with ATP where indicated. After a short incubation period, the reactions were analyzed by native PAGE. The RNase E(628–843)/RhlB/tRNA complex is stable in the presence of RraA (lane 5). However, upon addition of ATP, tRNA is released from the protein complex and replaced by RraA [*, lane 8, compare with identical complex of RNase E(628–843)/RhlB with RraA in lane 3]. Free RraA and tRNA comigrate with the buffer/dye front and, hence, RraA appears yellow, though there is no evidence for any interaction of RraA with RNA.

Surface charge of RraA and the suggested mechanism for participation of RraA in protein–RNA remodeling by RhlB. (A) Surface character of the Escherichia coli RraA (PDB code 1Q5X, Monzingo et al. 2003). Surface color represents electrostatic potential calculated by Pymol. (Top) The face of the RraA trimer exhibiting electronegative grooves radiating from the central hole along the monomer–monomer interface. The opposite face of the trimer is more neutral (bottom), and interacts with another trimer in the crystal (see Supplementary Material, Fig. 2). (B) Schematic of a possible mechanism of ATP-driven remodeling of RNase E(628–843)/RhlB/RNA complexes by RhlB. Positively charged, unstructured RNA-binding regions, such as RBD or AR2 in RNase E or the C-terminal extension of the helicase (denoted by positive charge marks) are able to bind RNA or RraA. In the case of a structured RNA, such as tRNA, the affinity for the nucleic acid is stronger than that for RraA. Addition of ATP causes remodeling of RNA interactions with the RNA-binding segments so that they can interact with RraA. It is possible that an intermediate containing both RraA and RNA is formed, but it must be comparatively unstable, since no super-shift could be observed in EMSA. There is no evidence whether all RNA-binding sites can simultaneously engage the same RraA trimer, but since any of them is sufficient for the remodeling and can bind to RraA, for simplicity the interactions with RraA or RNA are depicted as concurrent.