DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all processes involving RNA. They have nine conserved motifs that are required for ATP and RNA binding, and for linking phosphoanhydride cleavage of ATP with helicase activity. The Q motif is the most recently identified conserved element, and it occurs B17 amino acids upstream of motif I. There is a highly conserved, but isolated, aromatic group B17 amino acids upstream of the Q motif. These two elements are involved in adenine recognition and in ATPase activity of DEAD-box proteins. We made extensive analyses of the Q motif and upstream aromatic residue in the yeast translation-initiation factor Ded1. We made site-specific mutations and tested them for viability in yeast. Moreover, we purified various mutant proteins and obtained the Michaelis-Menten parameters for the ATPase activities. We also measured RNA affinities and strand-displacement activities. We find that the Q motif not only regulates ATP binding and hydrolysis but also regulates the affinity of the protein for RNA substrates and ultimately the helicase activity.

SF1 and SF2 helicases have structurally conserved cores containing seven to eight distinctive motifs and variable amino- and carboxyl-terminal flanking sequences. We have discovered a motif upstream of motif I that is unique to and characteristic of the DEAD box family of RNA helicases. It consists of a 9 amino acid sequence containing an invariant glutamine. A conserved phenylalanine occurs 17 aa further upstream. Sequence alignments, site-specific mutagenesis, and ATPase assays show that this motif and the upstream phenylalanine are highly conserved, that they are essential for viability in the yeast Saccharomyces cerevisiae, and that they control ATP binding and hydrolysis in the yeast translation-initiation factor eIF4A. These results are consistent with computer studies of the solved crystal structures.

We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding.The putative RNA helicases constitute a ubiquitous group of enzymes that are associated with all of the processes involving RNA, including transcription, ribosome biogenesis, mRNA splicing, RNA export, translation, and RNA degradation (reviewed in references 10, 30, 37, 42, and 46). They are characterized by two linked RecA-like domains (domains 1 and 2) that contain seven to eight conserved motifs (I, Ia, Ib, II, III, IV, V, and VI), and they are closely related to many of the DNA helicases (43). These RNA and DNA helicases are classified into either superfamily 1 (SF1) or superfamily 2 (SF2) according to the characteristics of the conserved motifs (17). Although some DNA and RNA helicases are known to have processive unwinding activity, most putative helicases show no or only weak activity in in vitro tests (46). Because contiguous double-stranded RNA is rare in vivo, RNA helicases may play a role primarily in RNA-RNA or RNA-protein remodeling (20,25,40,48,50) or as place setters to ensure the unidirectionality of complex reactions such as splicing or ribosomal biogenesis (46). Nevertheless, all of these family members are collectively known as helicases in the literature (17). Hence, we will use the term helicase in this paper as a generic term rather than as a functional description.The largest family of RNA helicases is the DEAD-box proteins of SF2 that typically have the sequence Asp-Glu-Ala-Asp (DEAD) in motif II (31). In addition to the eight conserved motifs, they contain three other features that are characteristic of this family: the upstream Q motif, which is involved in adenine recognition (11, 45), a conserved GG between motifs Ia and Ib (31), and a conserved QxxR, where x is any amino acid, bet...

Dbp6p is an essential putative ATP-dependent RNA helicase that is required for 60S-ribosomal-subunit assembly in the yeast Saccharomyces cerevisiae (D. Kressler, J. de la Cruz, M. Rojo, and P. Linder, Mol. Cell. Biol. 18:1855-1865, 1998). To identify factors that are functionally interacting with Dbp6p, we have performed a synthetic lethal screen with conditional dbp6 mutants. Here, we describe the cloning and the phenotypic analysis of the previously uncharacterized open reading frame YPL193W, which we renamed RSA1 (ribosome assembly 1). Rsa1p is not essential for cell viability; however, rsa1 null mutant strains display a slow-growth phenotype, which is exacerbated at elevated temperatures. The rsa1 null allele synthetically enhances the mild growth defect of weak dbp6 alleles and confers synthetic lethality when combined with stronger dbp6 alleles. Polysome profile analysis shows that the absence of Rsa1p results in the accumulation of half-mer polysomes. However, the pool of free 60S ribosomal subunits is only moderately decreased; this is reminiscent of polysome profiles from mutants defective in 60S-to-40S subunit joining. Pulse-chase labeling of pre-rRNA in the rsa1 null mutant strain indicates that formation of the mature 25S rRNA is decreased at the nonpermissive temperature. Interestingly, free 60S ribosomal subunits of a rsa1 null mutant strain that was grown for two generations at 37°C are practically devoid of the 60S-ribosomal-subunit protein Qsr1p/Rpl10p, which is required for joining of 60S and 40S subunits (D. P. Eisinger, F. A. Dick, and B. L. Trumpower, Mol. Cell. Biol. 17:5136-5145, 1997). Moreover, the combination of the ⌬rsa1 and qsr1-1 mutations leads to a strong synthetic growth inhibition. Finally, a hemagglutinin epitope-tagged Rsa1p localizes predominantly to the nucleoplasm. Together, these results point towards a function for Rsa1p in a late nucleoplasmic step of 60S-ribosomal-subunit assembly.The synthesis of ribosomes is one of the major cellular activities, which, in eukaryotes, takes place primarily, although not exclusively, in a specialized subnuclear compartment termed the nucleolus (33, 39). There, the ribosomal DNA is transcribed as precursors (pre-rRNAs), which undergo processing and covalent modification. Maturation of pre-rRNAs and their concomitant assembly with the ribosomal proteins (r-proteins) are dependent on various cis-acting elements and require a large number of nonribosomal trans-acting factors. Experimental evidence suggests that the basic outline of ribosome synthesis is conserved throughout eukaryotes. However, most of our knowledge comes from the combination of molecular genetics and biochemical approaches applied to the yeast Saccharomyces cerevisiae (reviewed in references 14, 46, 55, and 62).In S. cerevisiae, the large 60S ribosomal subunits are composed of 46 r-proteins and three rRNA species (5S, 5.8S, and 25S), while the small 40S ribosomal subunits contain 32 rproteins and the 18S rRNA (34, 62). Three of the four rRNAs (18S, 5.8S, and 25S) are transc...

Transport of lipids and proteins is a highly regulated process, which is required to maintain the integrity of various intracellular organelles in eukaryotic cells. Mutations along the yeast secretory pathway repress transcription of rRNA, tRNA, and ribosomal protein genes. Here, we show that these mutations also lead to a rapid and specific attenuation of translation initiation that occurs prior to the transcriptional inhibition of ribosomal components. Using distinct vesicular transport mutants and chlorpromazine, we have identified the eIF2alpha kinase Gcn2p and the eIF4E binding protein Eap1p as major mediators of the translation attenuation response. Finally, in chlorpromazine-treated cells, this response does not require Wsc1p or the protein kinase Pkc1p, both of which are upstream of the transcriptional repression of ribosomal components. Altogether, our results suggest that yeast cells not only evolved a transcriptional but also a translational control to assure efficient attenuation of protein synthesis when membranes are stressed.