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The newly discovered Q motif of DEAD-box RNA helicases regulates RNA-binding and helicase activity 总被引:7,自引:0,他引:7
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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
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The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis. 总被引:36,自引:6,他引:30
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eIF-4A is a eukaryotic translation initiation factor that is required for mRNA binding to ribosomes. It exhibits single-stranded RNA-dependent ATPase activity, and in combination with a second initiation factor, eIF-4B, it exhibits duplex RNA helicase activity. eIF-4A is the prototype of a large family of proteins termed the DEAD box protein family, whose members share nine highly conserved amino acid regions. The functions of several of these conserved regions in eIF-4A have previously been assigned to ATP binding, ATPase, and helicase activities. To define the RNA-binding region of eIF-4A, a UV-induced cross-linking assay was used to analyze binding of mutant eIF-4A proteins to RNA. Mutants carrying mutations in the ATP-binding region (AXXXXGKT), ATPase region (DEAD), helicase region (SAT), and the most carboxy-terminal conserved region of the DEAD family, HRIGRXXR, were tested for RNA cross-linking. We show that mutations, either conservative or not, in any one of the three arginines in the HRIGRXXR sequence drastically reduced eIF-4A cross-linking to RNA. In addition, all the mutations in the HRIGRXXR region abrogate RNA helicase activity. Some but not all of these mutations affect ATP binding and ATPase activity. This is consistent with the hypothesis that the HRIGRXXR region is involved in the ATP hydrolysis reaction and would explain the coupling of ATPase and RNA-binding/helicase activities. Our results show that the HRIGRXXR region, which is QRXGRXXR or QXXGRXXR in the RNA and DNA helicases of the helicase superfamily II, is involved in ATP hydrolysis-dependent RNA interaction during unwinding. We also show that mutations in other regions of eIF-4A that abolish ATPase activity sharply decrease eIF-4A cross-linking to RNA. A model is proposed in which eIF-4A first binds ATP, resulting in a change in eIF-4A conformation which allows RNA binding that is dependent on the HRIGRXXR region. Binding of RNA induces ATP hydrolysis, leading to a more stable interaction with RNA. This process is then linked to unwinding of duplex RNA in the presence of eIF-4B. 相似文献
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The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding. 总被引:13,自引:3,他引:10
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Vaccinia virus NPH-II is an essential nucleic acid-dependent nucleoside triphosphate that catalyzes unidirectional unwinding of duplex RNA containing a 3' tail. NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by several shared sequence motifs. The contribution of the conserved QRKGRVGRVNPG region to enzyme activity was assessed by alanine-scanning mutagenesis. Ten mutated versions of NPH-II were expressed in vaccinia virus-infected BSC-40 cells and purified by nickel affinity chromatography and glycerol gradient sedimentation. The mutated proteins were characterized with respect to RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Individual alanine substitutions at invariant residues Q-491, G-494, R-495, G-497, R-498, and G-502 caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. None of these mutations affected the binding of NPH-II to single-strand RNA or to the tailed duplex RNA used as a helicase substrate. Mutation of the strictly conserved position R-492 inhibited ATPase and helicase activities and also caused a modest decrement in RNA binding. Alanine mutations at the nonconserved position N-500 and the weakly conserved residue P-501 had no apparent effect on any activity associated with NPH-II, whereas a mutation at the weakly conserved position K-493 reduced helicase to one-third and ATPase to two-thirds of the activity of wild-type required for ATP hydrolysis and RNA unwinding but not for RNA binding. Because mutations in the HRxGRxxR motif of the prototypal DEAD box RNA helicase eIF-4A abolish or severely inhibit RNA binding, we surmise that the contribution of conserved helicase motifs to overall protein function is context dependent. 相似文献
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ATP hydrolysis activity of the DEAD box protein Rok1p is required for in vivo ROK1 function.
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The yeast ROK1 gene has been initially identified as a high copy plasmid suppressor of the kem1 null mutation and implicated in microtubule-mediated functions. Based on the deduced amino acid sequence of the ROK1 gene, Rok1p has been classified in the DEAD protein family of ATP-dependent RNA helicases. A subsequent report has suggested that Rok1p is required for rRNA processing. We report here the first study on the biochemical activity associated with Rok1p. The MBP-Rok1 hybrid protein was synthesized in Escherichia coli and purified by amylose affinity column and ion exchange chromatography. Rok1p has ATP hydrolysis activity. The significance of the conserved ATPase domains was addressed by generating a series of amino acid substitution mutations in these domains. Both in vivo lethality tests of the mutations and biochemical characterization of the mutant proteins suggest that ATP hydrolysis activity of Rok1p is essential for ROK1 function. The ATPase activity of Rok1p appears to be independent of single-stranded RNA. Furthermore, replacement of the first Arg in the HRIGR domain, the known RNA-binding domain, with Thr, Ile or Lys has no detectable effect on in vivo ROK1 function. The lack of RNA dependency and some of the mutational phenotypes of ROK1 differentiate this gene from other members of the family. 相似文献
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The identification of G-quadruplex (G4) binding proteins and insights into their mechanism of action are important for understanding the regulatory functions of G4 structures. Here, we performed an unbiased affinity-purification assay coupled with mass spectrometry and identified 30 putative G4 binding proteins from the fission yeast Schizosaccharomyces pombe. Gene ontology analysis of the molecular functions enriched in this pull-down assay included mRNA binding, RNA helicase activity, and translation regulator activity. We focused this study on three of the identified proteins that possessed putative arginine-glycine-glycine (RGG) domains, namely the Stm1 homolog Oga1 and the DEAD box RNA helicases Dbp2 and Ded1. We found that Oga1, Dbp2, and Ded1 bound to both DNA and RNA G4s in vitro. Both Dbp2 and Ded1 bound to G4 structures through the RGG domain located in the C-terminal region of the helicases, and point mutations in this domain weakened the G4 binding properties of the helicases. Dbp2 and Ded1 destabilized less thermostable G4 RNA and DNA structures, and this ability was independent of ATP but dependent on the RGG domain. Our study provides the first evidence that the RGG motifs in DEAD box helicases are necessary for both G4 binding and G4 destabilization. 相似文献
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Moncalian G Lengsfeld B Bhaskara V Hopfner KP Karcher A Alden E Tainer JA Paull TT 《Journal of molecular biology》2004,335(4):937-951
The repair of double-strand breaks in DNA is an essential process in all organisms, and requires the coordinated activities of evolutionarily conserved protein assemblies. One of the most critical of these is the Mre11/Rad50 (M/R) complex, which is present in all three biological kingdoms, but is not well-understood at the biochemical level. Previous structural analysis of a Rad50 homolog from archaebacteria illuminated the catalytic core of the enzyme, an ATP-binding domain related to the ABC transporter family of ATPases. Here, we present the crystallographic structure of the Rad50 mutant S793R. This missense signature motif mutation changes the key serine residue in the signature motif that is conserved among Rad50 homologs and ABC ATPases. The S793R mutation is analogous to the mutation S549R in the cystic fibrosis transmembrane conductance regulator (CFTR) that results in cystic fibrosis. We show here that the serine to arginine change in the Rad50 protein prevents ATP binding and disrupts the communication among the other ATP-binding loops. This structural change, in turn, alters the communication between Rad50 monomers and thus prevents Rad50 dimerization. The equivalent mutation was made in the human Rad50 gene, and the resulting mutant protein did form a complex with Mre11 and Nbs1, but was specifically deficient in all ATP-dependent enzymatic activities. This signature motif structure-function homology extends to yeast, because the same mutation introduced into the Saccharomyces cerevisiae RAD50 gene generated an allele that failed to complement a rad50 deletion strain in DNA repair assays in vivo. These structural and biochemical results extend our understanding of the Rad50 catalytic domain and validate the use of the signature motif mutant to test the role of Rad50 ATP binding in diverse organisms. 相似文献
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Biswas I Obmolova G Takahashi M Herr A Newman MA Yang W Hsieh P 《Journal of molecular biology》2001,305(4):805-816
The DNA mismatch repair protein, MutS, is a dimeric protein that recognizes mismatched bases and has an intrinsic ATPase activity. Here, a series of Taq MutS proteins having C-terminal truncations in the vicinity of a highly conserved helix-u-turn-helix (HuH) motif are assessed for subunit oligomerization, ATPase activity and DNA mismatch binding. Those proteins containing an intact HuH region are dimers; those without the HuH region are predominantly monomers in solution. Steady-state kinetics of truncated but dimeric MutS proteins reveals only modest decreases in their ATPase activity compared to full-length protein. In contrast, disruption of the HuH region results in a greatly attenuated ATPase activity. In addition, only dimeric MutS proteins are proficient for mismatch binding. Finally, an analysis of the mismatch repair competency of truncated Escherichia coli MutS proteins in a rifampicin mutator assay confirms that the HuH region is critical for in vivo function. These findings indicate that dimerization is critical for both the ATPase and DNA mismatch binding activities of MutS, and corroborate several key features of the MutS structure recently deduced from X-ray crystallographic studies. 相似文献
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Active DNA-dependent ATPase A Domain (ADAAD) is a SWI2/SNF2 protein that hydrolyzes ATP in the presence of stem-loop DNA that contains both double-stranded and single-stranded regions. ADAAD possesses the seven helicase motifs that are a characteristic feature of all the SWI2/SNF2 proteins present in yeast as well as mammalian cells. In addition, these proteins also possess the Q motif ~17 nucleotides upstream of motif I. Using site-directed mutagenesis, we have sought to define the role of motifs Q and I in ATP hydrolysis mediated by ADAAD. We show that in ADAAD both motifs Q and I are required for ATP catalysis but not for ATP binding. In addition, the conserved glutamine present in motif Q also dictates the catalytic rate. The ability of the conserved glutamine present in motif Q to dictate the catalytic rate has not been observed in helicases. Further, the SWI2/SNF2 proteins contain a conserved glutamine, one amino acid residue downstream of motif I. This conserved glutamine, Q244 in ADAAD, also directs the rate of catalysis but is not required either for hydrolysis or for ligand binding. Finally, we show that the adenine moiety of ATP is sufficient for interaction with SWI2/SNF2 proteins. The γ-phosphate of ATP is required for inducing the conformational change that leads to ATPase activity. Thus, the SWI2/SNF2 proteins despite sequence conservation with helicases appear to behave in a manner distinct from that of the helicases. 相似文献
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DEAD box family helicases consist of a helicase core that is formed by two flexibly linked RecA-like domains. The helicase activity can be regulated by N- or C-terminal extensions flanking the core. Thermus thermophilus heat resistant RNA-dependent ATPase (Hera) is the first DEAD box helicase that forms a dimer using a unique dimerization domain. In addition to the dimerization domain, Hera contains a C-terminal RNA binding domain (RBD) that shares sequence homology only to uncharacterized proteins of the Deinococcus/Thermus group. The crystal structure of Hera_RBD reveals the fold of an altered RNA recognition motif (RRM) with limited structural homology to the RBD of the DEAD box helicase YxiN from Bacillus subtilis. Comparison with RRM/RNA complexes shows that a RNA binding mode different than that suggested for YxiN, but similar to U1A, can be inferred for Hera. The orientation of the RBD relative to the helicase core was defined in a second crystal structure of a Hera fragment including the C-terminal RecA domain, the dimerization domain, and the RBD. The structures allow construction of a model for the entire Hera helicase dimer. A likely binding surface for large RNA substrates that spans both RecA-like domains and the RBD is identified. 相似文献
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The Rad51 recombinase polymerizes on ssDNA to yield a right-handed nucleoprotein filament, called the presynaptic filament, that can search for homology in duplex DNA and pair the recombining DNA molecules to form a DNA joint. ATP is needed for presynaptic filament assembly and homologous DNA pairing, but the roles of ATP binding and ATP hydrolysis in the overall reaction scheme have not yet been clearly defined. To address this issue, we have constructed two mutants of hRad51, hRad51 K133A and hRad51 K133R, expressed these mutant variants in Escherichia coli, and purified them to near homogeneity. Both hRad51 mutant variants are greatly attenuated for ATPase activity, but hRad51 K133R retains the ability to protect DNA from restriction enzyme digest and induce topological changes in duplex DNA in an ATP-dependent manner, whereas the hRad51 K133A variant is inactive. With biochemical means, we show that the presynaptic filament becomes greatly stabilized when ATP hydrolysis is prevented, leading to an enhanced ability of the presynaptic filament to catalyze homologous pairing. These results help form the basis for understanding the functions of ATP binding and ATP hydrolysis in hRad51-mediated recombination reactions. 相似文献
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Alícia Broto Luca Martinelli Enrique Querol Jaume Piñol Ignacio Fita 《Molecular microbiology》2012,86(2):382-393
Mycoplasma genitalium is an emerging human pathogen with the smallest genome found among self‐replicating organisms. M. genitalium presents a complex cytoskeleton with a differentiated protrusion known as the terminal organelle. This polar structure plays a central role in functions essential for the virulence of the microorganism, such as motility and cell‐host adhesion. A well‐conserved Enriched in Aromatic and Glycine Residues motif, the EAGR box, is present in many of the proteins found in the terminal organelle. We determined the crystal structure of the globular domain from M. genitalium MG200 that contains an EAGR box. This structural information is the first at near atomic resolution for the components of the terminal organelle. The structure revealed a dimer stabilized by a compact hydrophobic core that extends throughout the dimer interface. Monomers present a new fold that contains an accurate intra‐subunit symmetry relating two conspicuous hairpins. Some features, such as the domain plasticity and the presence and organization of the intra‐ and inter‐subunit symmetry axes, support a role for the EAGR box in protein–protein interactions. Genetic, biochemical and microcinematography analyses of MG200 variants lacking the EAGR box containing domain confirm the relevant and specific association of this domain with cell motility. 相似文献
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Transformation of MutL by ATP binding and hydrolysis: a switch in DNA mismatch repair 总被引:26,自引:0,他引:26
The MutL DNA mismatch repair protein has recently been shown to be an ATPase and to belong to an emerging ATPase superfamily that includes DNA topoisomerase II and Hsp90. We report here the crystal structures of a 40 kDa ATPase fragment of E. coli MutL (LN40) complexed with a substrate analog, ADPnP, and with product ADP. More than 60 residues that are disordered in the apoprotein structure become ordered and contribute to both ADPnP binding and dimerization of LN40. Hydrolysis of ATP, signified by subsequent release of the gamma-phosphate, releases two key loops and leads to dissociation of the LN40 dimer. Dimerization of the LN40 region is required for and is the rate-limiting step in ATP hydrolysis by MutL. The ATPase activity of MutL is stimulated by DNA and likely acts as a switch to coordinate DNA mismatch repair. 相似文献
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DEAD box helicases use the energy of ATP hydrolysis to remodel RNA structures or RNA/protein complexes. They share a common helicase core with conserved signature motifs, and additional domains may confer substrate specificity. Identification of a specific substrate is crucial towards understanding the physiological role of a helicase. RNA binding and ATPase stimulation are necessary, but not sufficient criteria for a bona fide helicase substrate. Here, we report single molecule FRET experiments that identify fragments of the 23S rRNA comprising hairpin 92 and RNase P RNA as substrates for the Thermus thermophilus DEAD box helicase Hera. Both substrates induce a switch to the closed conformation of the helicase core and stimulate the intrinsic ATPase activity of Hera. Binding of these RNAs is mediated by the Hera C-terminal domain, but does not require a previously proposed putative RNase P motif within this domain. ATP-dependent unwinding of a short helix adjacent to hairpin 92 in the ribosomal RNA suggests a specific role for Hera in ribosome assembly, analogously to the Escherichia coli and Bacillus subtilis helicases DbpA and YxiN. In addition, the specificity of Hera for RNase P RNA may be required for RNase P RNA folding or RNase P assembly. 相似文献
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囊性纤维化跨膜电导调节体(cystic fibrosis transmembrane conductance regulator,CFTR)是一种Cl^-通道,属于ATP结合(ATP-binding cassette,ABC)转运体超家族。CFTR功能缺陷是高加索人种中普遍存在的致死性常染色体隐性遗传疾病囊性纤维化(cystic fibrosis,CF)发生的主要原因。这种疾病患者各组织上皮细胞内Cl^-转运失调。目前,与CF相关的不同突变超过1400种。CFTR调节(regulatory,R)域负责调控,核苷酸结合域(nucleotide-binding domains,NBDs)NBD1和NBD2负责ATP结合和水解门控。近期研究发现CFFR的NBDs与其它ABC蛋白一样可以二聚化。二聚化过程中,NBD1和NBD2首-尾相连,一个NBD上的WalkerA和B模块与另一个NBD提供的标签序列(signature sequence)形成ATP结合袋(ATP-binding pockets,ABPs)ABP1和ABP2。ABPs中与ATP结合相关的氨基酸突变实验揭示,ABP1和ABP2在CFTR的ATP依赖门控中发挥不同作用。ABP2由NBD2上的WalkA和B模块与NBD1提供的标签序列形成,它与ATP结合催化通道开放,而ABP1单独与ATP结合不能促进通道开放,只能稳定通道构象。有一些CFrR突变相关疾病的特征就是门控失调,进一步深入研究CFTR的NBD1和NBD2如何通过相互作用而达到通道门控,将为药理学研究提供更多所需的机制信息,有利于为CF治疗的药物设计铺平道路。 相似文献
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