SummaryRibosome assembly in Escherichia coli involves 54 ribosomal proteins and three RNAs. Whereas functional subunits can be reconstituted in vitro from the isolated components, this process requires long incubation times and high temperatures compared with the in vivo situation, suggesting that non-ribosomal factors facilitate assembly in vivo . Here, we show that SrmB, a putative DEAD-box RNA helicase, is involved in ribosome assembly. The deletion of the srmB gene causes a slow-growth phenotype at low temperature. Polysome profile analyses of the corresponding cells reveal a deficit in free 50S ribosomal subunits and the accumulation of a new particle sedimenting around 40S. Analysis of the ribosomal RNA and protein contents of the 40S particle indicates that it represents a large subunit that is incompletely assembled. In particular, it lacks L13, one of the five ribosomal proteins that are essential for the early assembly step in vitro . Sucrose gradient fractionation also shows that, in wild-type cells, SrmB associates with a pre50S particle. From our results, we propose that SrmB is involved in an early step of 50S assembly that is necessary for the binding of L13. This step may consist of a structural rearrangement that, at low temperature, cannot occur without the assistance of this putative RNA helicase.

SummaryRNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay. While the C-terminal half of this 1061-residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into à degradosome', only the N-terminal half, which carries the catalytic activity, is required for growth. We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E. This mutation resembles the N-terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected. Another mutation (rne105 ) removing the last 469 residues behaves similarly. Thus, the C-terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA. A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne pnp mutants. All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization. Arti®cial mRNAs generated by E. coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth.

The Ded1 protein (Ded1p), a member of the DEAD-box family, has recently been shown to be essential for translation initiation in Saccharomyces cerevisiae. Here, we show that Ded1p purified from Escherichia coli has an ATPase activity, which is stimulated by various RNA substrates. Using an RNA strand-displacement assay, we show that Ded1p has also an ATP-dependent RNA unwinding activity. Hydrolysis of ATP is required for this activity: the replacement of ATP by a nonhydrolyzable analog or a mutation in the DEAD motif abolishing ATPase activity results in loss of RNA unwinding. We find that cells harboring a Ded1 protein with this mutated DEAD motif are nonviable, suggesting that the ATPase and RNA helicase activities of this protein are essential to the cell. Finally, RNA binding measurements indicate that the presence of ATP, but not ADP, increases the affinity of Ded1p for duplex versus singlestranded RNA; we discuss how this differential effect might drive the unwinding reaction.DEAD-box proteins form a large family of putative RNA helicases that show sequence similarity to eIF4A, a eukaryotic translation initiation factor. All members of the DEAD-box family share eight conserved amino acid motifs, including the characteristic sequence Asp-Glu-Ala-Asp (DEAD in the singleletter code) that inspired their name (1). Sequence comparisons identified the closely related DEAH-and DExH-box families, which together with the DEAD-box proteins, form the helicase superfamily II (2). The DEAH and DExH families notably include DNA helicases involved in DNA replication and recombination. The putative RNA helicases of superfamily II are found in a wide range of organisms, including bacteria, viruses, and eukaryotes ranging from yeast to humans. Although they are involved in very diverse cellular functions, such as pre-mRNA splicing, rRNA processing, and mRNA export, translation, and decay, they are all supposed to share in common an RNA helicase activity (3, 4). This activity has been inferred from the ability of eIF4A to melt out mRNA structure (5) or to dissociate an RNA duplex in vitro (6), in an ATP-dependent manner. However, although NTPase activity has been demonstrated for all purified DEAD-box and related proteins, RNA helicase activity has been characterized for only a few of them and remains conjectural in most cases.Nevertheless, numerous steps of gene expression are likely to require RNA helicase activity, either to unwind RNA secondary structures or to rearrange large RNA structures, or even to disrupt RNA-protein interactions. For example, transient base pairings between small nuclear RNAs and between small nuclear RNAs and pre-mRNA, which occur during pre-mRNA splicing, are often mutually exclusive and thus need to form and dissociate sequentially. At least eight DEAD-box and related proteins have thus far been shown to be required for splicing in yeast and may accomplish these structural rearrangements (7). Similarly, 13 DEAD-box are assumed to be involved in extensive rearrangements between pre-rRNA and ribos...