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1.
Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a “helicase” strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (kcat), but not the affinity for ATP (Km). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of γ-PO4 of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the β,γ-phosphoanhydride bond of ATP.  相似文献   

2.
3.
The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions—one the previously noted post II region in the helicase core and the other in the CTE—that may help displace or sequester the opposite RNA strand during RNA unwinding.  相似文献   

4.
The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are general RNA chaperones that function in splicing mitochondrial group I and group II introns and in translational activation. Both proteins consist of a conserved ATP-dependent RNA helicase core region linked to N and C-terminal domains, the latter with a basic tail similar to many other DEAD-box proteins. In CYT-19, this basic tail was shown to contribute to non-specific RNA binding that helps tether the core helicase region to structured RNA substrates. Here, multiple sequence alignments and secondary structure predictions indicate that CYT-19 and Mss116p belong to distinct subgroups of DEAD-box proteins, whose C-terminal domains have a defining extended α-helical region preceding the basic tail. We find that mutations or C-terminal truncations in the predicted α-helical region of Mss116p strongly inhibit RNA-dependent ATPase activity, leading to loss of function in both translational activation and RNA splicing. These findings suggest that the α-helical region may stabilize and/or regulate the activity of the RNA helicase core. By contrast, a truncation that removes only the basic tail leaves high RNA-dependent ATPase activity and causes only a modest reduction in translation and RNA splicing efficiency in vivo and in vitro. Biochemical analysis shows that deletion of the basic tail leads to weaker non-specific binding of group I and group II intron RNAs, and surprisingly, also impairs RNA-unwinding at saturating protein concentrations and nucleotide-dependent tight binding of single-stranded RNAs by the RNA helicase core. Together, our results indicate that the two sub-regions of Mss116p's C-terminal domain act in different ways to support and modulate activities of the core helicase region, whose RNA-unwinding activity is critical for both the translation and RNA splicing functions.  相似文献   

5.
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 approximately 17 amino acids upstream of motif I. There is a highly conserved, but isolated, aromatic group approximately 17 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.  相似文献   

6.
The putative RNA helicases of the DEAD-box protein family are involved in pre-mRNA splicing, rRNA maturation, ribosome assembly, and translation. Members of this protein family have been identified in organisms from Escherichia coli to humans, but except for the translation initiation factor 4A, there have been no reports on the characterization of other DEAD-box proteins from plants. Here we report on a novel member of the DEAD-box protein family, the plant RNA helicase 75 (PRH75). PRH75 is localized in the nucleus and contains two domains for RNA binding. One is located at the C terminus and is similar to RGG RNA-binding domains of nucleus-localized RNA-binding proteins. The other one is located between amino acids 308 and 622, a region containing the conserved motif VI characteristic of DEAD-box proteins and known as the RNA-binding site of eIF-4A. The N-terminal 81 amino acids are sufficient for nuclear targeting of the protein. Northern and Western blot analyses show that PRH75 is mainly expressed in young and rapidly developing tissues. The purified recombinant PRH75 has a weak ATPase activity which is barely stimulated by RNA ligands. The fractionation of spinach whole-cell extracts by glycerol gradient centrifugation and gel filtration on a Superdex 200 column shows that the protein exists in a complex of about 500 kDa. Possible biological functions of PRH75 as well as structure-function relationships in the context of its modular primary structure are discussed.  相似文献   

7.
DEAD-box proteins are RNA-dependent ATPase enzymes that have been implicated in nearly all aspects of RNA metabolism. Since many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, to bind nucleic acid, and to promote helix unwinding, DEAD-box proteins are generally thought to modulate RNA structure in vivo. However, the extent to which these enzymatic properties are important for the in vivo functions of DEAD-box proteins remains unclear. To evaluate how these properties influence DEAD-box protein native function, we probed the importance of several highly conserved residues in the yeast DEAD-box protein Mss116p, which is required for the splicing of all mitochondrial catalytic introns in Saccharomyces cerevisiae. Using an MSS116 deletion strain, we have expressed plasmid-borne variants of MSS116 containing substitutions in residues predicted to be important for extensive networks of interactions required for ATP hydrolysis and helix unwinding. We have analyzed the importance of these residues to the splicing functions of Mss116p in vivo and compared these results with the biochemical properties of recombinant proteins determined here and in previously published work. We observed that the efficiency by which an Mss116p variant catalyzes ATP hydrolysis correlates with facilitating mitochondrial splicing, while efficient helix unwinding appears to be insufficient for splicing. In addition, we show that each splicing-defective variant affects the splicing of structurally diverse introns to the same degree. Together, these observations suggest that the efficiency by which Mss116p catalyzes the hydrolysis of ATP is critical for all of its splicing functions in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, these observations support a model in which enzyme turnover is a crucial factor in Mss116p splicing function. These results are discussed in the context of current models of Mss116p-facilitated splicing.  相似文献   

8.
DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.  相似文献   

9.
Specific interactions of transmembrane helices play a pivotal role in the folding and oligomerization of integral membrane proteins. The helix-helix interfaces frequently depend on specific amino acid patterns. In this study, a heptad repeat pattern was randomized with all naturally occurring amino acids to uncover novel sequence motifs promoting transmembrane domain interactions. Self-interacting transmembrane domains were selected from the resulting combinatorial library by means of the ToxR/POSSYCCAT system. A comparison of the amino acid composition of high-and low-affinity sequences revealed that high-affinity transmembrane domains exhibit position-specific enrichment of histidine. Further, sequences containing His preferentially display Gly, Ser, and/or Thr residues at flanking positions and frequently contain a C-terminal GxxxG motif. Mutational analysis of selected sequences confirmed the importance of these residues in homotypic interaction. Probing heterotypic interaction indicated that His interacts in trans with hydroxylated residues. Reconstruction of minimal interaction motifs within the context of an oligo-Leu sequence confirmed that His is part of a hydrogen bonded cluster that is brought into register by the GxxxG motif. Notably, a similar motif contributes to self-interaction of the BNIP3 transmembrane domain.  相似文献   

10.
Eukaryotic gene expression involves numerous biochemical steps that are dependent on RNA structure and ribonucleoprotein (RNP) complex formation. The DEAD-box class of RNA helicases plays fundamental roles in formation of RNA and RNP structure in every aspect of RNA metabolism. In an effort to explore the diversity of biological roles for DEAD-box proteins, our laboratory recently demonstrated that the DEAD-box protein Dbp2 associates with actively transcribing genes and is required for normal gene expression in Saccharomyces cerevisiae. We now provide evidence that Dbp2 interacts genetically and physically with the mRNA export factor Yra1. In addition, we find that Dbp2 is required for in vivo assembly of mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A)+ RNA. Strikingly, we also show that Dbp2 is an efficient RNA helicase in vitro and that Yra1 decreases the efficiency of ATP-dependent duplex unwinding. We provide a model whereby messenger ribonucleoprotein (mRNP) assembly requires Dbp2 unwinding activity and once the mRNP is properly assembled, inhibition by Yra1 prevents further rearrangements. Both Yra1 and Dbp2 are conserved in multicellular eukaryotes, suggesting that this constitutes a broadly conserved mechanism for stepwise assembly of mature mRNPs in the nucleus.  相似文献   

11.
12.
DEAD-box proteins are found in all domains of life and participate in almost all cellular processes that involve RNA. The presence of DEAD and Helicase_C conserved domains distinguish these proteins. DEAD-box proteins exhibit RNA-dependent ATPase activity in vitro, and several also show RNA helicase activity. In this study, we analyzed the distribution and architecture of DEAD-box proteins among bacterial genomes to gain insight into the evolutionary pathways that have shaped their history. We identified 1,848 unique DEAD-box proteins from 563 bacterial genomes. Bacterial genomes can possess a single copy DEAD-box gene, or up to 12 copies of the gene, such as in Shewanella. The alignment of 1,208 sequences allowed us to perform a robust analysis of the hallmark motifs of DEAD-box proteins and determine the residues that occur at high frequency, some of which were previously overlooked. Bacterial DEAD-box proteins do not generally contain a conserved C-terminal domain, with the exception of some members that possess a DbpA RNA-binding domain (RBD). Phylogenetic analysis showed a separation of DbpA-RBD-containing and DbpA-RBD-lacking sequences and revealed a group of DEAD-box protein genes that expanded mainly in the Proteobacteria. Analysis of DEAD-box proteins from Firmicutes and γ-Proteobacteria, was used to deduce orthologous relationships of the well-studied DEAD-box proteins from Escherichia coli and Bacillus subtilis. These analyses suggest that DbpA-RBD is an ancestral domain that most likely emerged as a specialized domain of the RNA-dependent ATPases. Moreover, these data revealed numerous events of gene family expansion and reduction following speciation.  相似文献   

13.
Helicases are motor proteins of biological system, which catalyze the opening of energetically stable duplex nucleic acids in an ATP-dependent manner and thereby are involved in almost all aspects of nucleic acid metabolism including cell cycle progression. They contain several conserved domains including the DEAD-box and also several unique domains associated with these. The Pfam database (http://pfam.janelia.org/) is a large collection of protein families, each represented by multiple sequence alignments and hidden Markov models (HMMs). A diverse range of proteins are found in nature, and the functional specificity to each protein, to a greater extent, is imparted by its domain architecture. To this extent, a DEAD-box ATP-dependent RNA helicase (LOC_Os01g36890; Genomic sequence length: 6284 nucleotides; CDS length: 1299 nucleotides; Protein length: 432 amino acids) was studied. The protein sequence was imported for domain search on Pfam. This particular Pfam entry after covering a large proportion of the sequences in the underlying database has generated a more comprehensive coverage across a wide range of phyla of the known domains that are associated with the typical DEAD-box helicase motif. A total of 362 domain architectures were recollected from the Pfam database for the Family: DEAD (PF00270). We have therefore systematically analyzed the domains closely associated with DEAD-motif, which occur in a variety of proteins and can provide insights into their function.  相似文献   

14.
The mammalian SPRY domain- and SOCS box-containing proteins, SPSB1 to SPSB4, belong to the SOCS box family of E3 ubiquitin ligases. Substrate recognition sites for the SPRY domain are identified only for human Par-4 (ELNNNL) and for the Drosophila orthologue GUSTAVUS binding to the DEAD-box RNA helicase VASA (DINNNN). To further investigate this consensus motif, we determined the crystal structures of SPSB1, SPSB2, and SPSB4, as well as their binding modes and affinities for both Par-4 and VASA. Mutation of each of the three Asn residues in Par-4 abrogated binding to all three SPSB proteins, while changing EL to DI enhanced binding. By comparison to SPSB1 and SPSB4, the more divergent protein SPSB2 showed only weak binding to Par-4 and was hypersensitive to DI substitution. Par-4(59-77) binding perturbed NMR resonances from a number of SPSB2 residues flanking the ELNNN binding site, including loop D, which binds the EL/DI sequence. Although interactions with the consensus peptide motif were conserved in all structures, flanking sites in SPSB2 were identified as sites of structural change. These structural changes limit high-affinity interactions for SPSB2 to aspartate-containing sequences, whereas SPSB1 and SPSB4 bind strongly to both Par-4 and VASA peptides.  相似文献   

15.
在RNA代谢过程中,需要许多蛋白和核酸的参与,其中一类蛋白就是RNA解旋酶。RNA解旋酶通过水解ATP获得能量来参与RNA代谢的多个方面,包括核内转录、pre-mRNA的剪切、核糖体发生、核质运输、蛋白质翻译、RNA降解、细胞器内基因的表达。DEAD-box蛋白家族是RNA解旋酶中最大的亚家族,它具有9个保守结构域,因motifyⅡ的保守氨基酸序列Asp-Glu-Ala-Asp(DEAD)而命名。该家族在酵母、拟南芥(Arabidopsis thaliana Heynh.)和人类基因组中都有较多的家庭成员。近年来,研究者对拟南芥DEAD-box蛋白家族的结构和功能进行了一些研究,本文着重总结DEAD-box基因家族对拟南芥生长发育的影响。  相似文献   

16.
17.
Since our characterization of the slit cDNA sequence, encoding a protein secreted by glial cells and involved in the formation of axonal pathways in Drosophila, we have discovered that the protein contains two additional sequence motifs that are highly conserved in a variety of proteins. A search of the GenPept database with the 73 amino acids at the carboxy terminus of slit revealed that this region contains significant similarity to a carboxy-terminal domain found in six other exported proteins. This observation has allowed us to define a new carboxy-terminal protein motif. In addition, comparisons with a 202 amino acid domain residing between epidermal growth factor (EGF) repeats in slit shows this region to be conserved in laminin, agrin and perlecan and, strikingly, also to lie between EGF repeats in both agrin and perlecan. Our analysis suggests this motif is involved in mediating interactions among extracellular proteins. Consistent with our previous characterization of the slit protein, both new motifs are found only in extracellular proteins. The identification of these two conserved motifs in slit reveals that the entire 1469 amino acids of the protein are made up of modular regions similar to those conserved in other extracellular proteins.  相似文献   

18.
For type I restriction systems, recently determined nucleotide sequences predict conserved amino acids in the subunit that is essential for restriction but not modification (HsdR). The conserved sequences emphasize motifs characteristic of the DEAD-box family of proteins which comprises putative helicases, and they identify a new candidate for motif IV. We provide evidence based on an analysis of Eco KI which supports both the relevance of DEAD-box motifs to the mechanism of restriction and the new definition of motif IV. Amino acid substitutions within the newly identified motif IV and those in six other previously identified DEAD-box motifs, but not in the original motif IV, confer restriction-deficient phenotypes. We have examined the relevance of the DEAD-box motifs to the restriction pathway by determining the steps permitted in vitro by the defective enzymes resulting from amino acid substitutions in each of the seven motifs. Eco KI purified from the seven restriction-deficient mutants binds to an unmethylated target sequence and, in the presence of AdoMet, responds to ATP by undergoing the conformational change essential for the pathway of events leading to DNA cleavage. The seven enzymes have little or no ATPase activity and no endonuclease activity, but they retain the ability to nick unmodified DNA, though at reduced rates. Nicking of a DNA strand could therefore be an essential early step in the restriction pathway, facilitating the ATP-dependent translocation of DNA, particularly if this involves DNA helicase activity.  相似文献   

19.
RNA helicases of the DExD/H-box superfamily are critically involved in all RNA-related processes. No crystal structures of human DExH-box domains had been determined previously, and their structures were difficult to predict owing to the low level of homology among DExH-motif-containing proteins from diverse species. Here we present the crystal structures of the conserved domain 1 of the DEIH-motif-containing helicase DHX9 and of the DEAD-box helicase DDX20. Both contain a RecA-like core, but DHX9 differs from DEAD-box proteins in the arrangement of secondary structural elements and is more similar to viral helicases such as NS3. The N-terminus of the DHX9 core contains two long α-helices that reside on the surface of the core without contributing to nucleotide binding. The RNA-polymerase-II-interacting minimal transactivation domain sequence forms an extended loop structure that resides in a hydrophobic groove on the surface of the DEIH domain. DHX9 lacks base-selective contacts and forms an unspecific but important stacking interaction with the base of the bound nucleotide, and our biochemical analysis confirms that the protein can hydrolyze ATP, guanosine 5′-triphosphate, cytidine 5′-triphosphate, and uridine 5′-triphosphate. Together, these findings allow the localization of functional motifs within the three-dimensional structure of a human DEIH helicase and show how these enzymes can bind nucleotide with high affinity in the absence of a Q-motif.  相似文献   

20.
Linder P 《Nucleic acids research》2006,34(15):4168-4180
DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family.  相似文献   

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