Summary of structures and properties of the five DEAD-box proteins from E.coli. Regions encompassing conserved residues (i.e. helicase core) are shown as open rectangles; stripes denote the positions of the conserved motifs (Q to VI), whereas the N- and C-terminal extensions (drawn to scale) are shown as thin lines. Also indicated are the proposed functions of the proteins, their in vitro activity (+ means that the protein is able to dissociate an RNA duplex in an ATP-dependent manner), and the phenotype associated with the deletion of the gene (no, no visible growth defect; cs, cold-sensitive growth).

(A) Distribution of putative orthologues of the E.coli DEAD-box proteins over the bacterial phylogenic tree, as revealed by BLAST analysis. The whole sequence of the proteins (coloured rectangles) or, for DbpA, the C-terminal extension (open rectangles; see Figure 1 and text for details) have been used as probes. The tree was adapted from a web page of The National Center for Biotechnology Information, National Library of Medicine, USA (). For a given DEAD-box protein (see inset for the colour code) and for each phylum, class or order, the height of the histogram is proportional to the highest score observed for a representative of this phylum, class or order. The scores obtained with the whole sequence probes have been corrected to take into account the homology that exists between unrelated DEAD-box proteins (see text). Red figures (italics) correspond to the number of sequences in the databank. Note that eventually bacteria distant from E.coli contain other DEAD-box helicases whose sequence are unrelated to those of E.coli and therefore have not been detected here. The mention ‘S’ or ‘L’ following the histogram refers to RNase E, the genuine partner of RhlB in E.coli. ‘S’ (Short) means that all identified RNase E orthologues consist of the catalytic region only, whereas ‘L’ (Long) means that at least one RNase E representative carries an extension of >100 residues with no homology to the catalytic core of E.coli RNase E (first 500 residues). Such extension could act as a scaffold for the assembly of the degradosome. The subscript (1–4) refers to the position of the extension with respect to the catalytic core (see B). (B) Schematic drawing showing the different configurations of RNase E observed in the phylogenetic tree.

Models for the unwinding activity of E.coli DEAD-box proteins. (A) Substrate recognition. The helicase is shown in red, with the core RecA-like domains and the C-terminal domain appearing as closed and open circles, respectively. The helicase core is assumed to bind to one strand of the RNA duplex, as observed with the DEAD-box protein Vasa in the presence of ATP (6); this strand is shown in light green, with the complementary strand in dark green. Moreover, the relative orientations of the core and bound RNA are assumed to be the same as in Vasa, i.e. the N-terminal domain (N) interacts with the 3′ side of the RNA. For the sake of simplicity, putative secondary binding sites (SrmB, CsdA, RhlB) are assumed to bind the same strand as the core, although this is not necessarily the case [cf DbpA, d; (23)]. (i) RhlE can unwind blunt end duplexes, and therefore its core presumably interacts directly with one strand of the duplex. (ii) SrmB and CsdA require single-stranded extensions, but these extensions can be either 5′ (left) or 3′ (right) to the duplex. We propose that in this case also the core interacts directly with one strand of the duplex, and that the extensions are used not for translocation but for binding the helicase via a secondary RNA binding site, which, by analogy with DbpA (see below), is shown here within the C-terminal domain. (iii) RhlB is inactive as a helicase unless it binds RNase E or a fragment thereof (in blue) carrying an arginine-rich region. This fragment may constitute a secondary RNA binding site that interacts with single-stranded extensions, as proposed above. The RhlB region that contacts RNase E has not been determined; it is assumed here to lie in the C-terminal domain. (iv) The DbpA C-terminal domain binds tightly to helix 92 (H92) of 23S rRNA, and the protein can unwind duplexes that are located either 3′ (a) or 5′ (b) to the helix. DbpA is unique amongst E.coli DEAD-box helicases in strictly requiring a 3′ single-stranded extension: substrates lacking such extension cannot be unwound (c). However, this extension does not need to lie on the same strand as helix 92 (d). (B) Model explaining why most DEAD-box helicases are poorly processive and do not require a single-stranded extension of definite polarity [based on (6)]. In the presence of ATP, the protein binds tightly to RNA, which can be either single- or double-stranded. However, the presence of a ‘wedge’ (white triangle) encompassing motif Ib (Figure 1) induces a kink in the bound RNA that is locally incompatible with double strandedness. Therefore, bound duplexes unwind over a few bases. The protein may preferentially bind near the duplex ends so that thermal fraying assists unwinding. After ATP hydrolysis, the partially unwound duplex is free to dissociate (‘unstable duplex’) or reanneal (‘stable duplex’), depending upon its stability. At this stage, the protein may either dissociate, or remain RNA-bound as illustrated here.