Yeast RNA helicases of the DEAD-box family involved in translation initiation

RNA helicases of the DEAD-box and related families have been found to be required for all processes involving RNA molecules. Biochemical and genetic analyses have shown that at least two RNA helicases are required for translation initiation in yeast. Although it is generally believed that these enzymes are necessary to unwind secondary structures in the 5' untranslated region of mRNAs, their exact role has not been convincingly shown. We discuss here our present knowledge of the function of eIF4A and Ded1p, two DEAD-box proteins required for translation in eukaryotic cells.     

The Ded1/DDX3 subfamily of DEAD-box RNA helicases

Abstract

In eukaryotic organisms, the orthologs of the DEAD-box RNA helicase Ded1p from yeast and DDX3 from human form a well-defined subfamily that is characterized by high sequence conservation in their helicase core and their N- and C- termini. Individual members of this Ded1/DDX3 subfamily perform multiple functions in RNA metabolism in both nucleus and cytoplasm. Ded1/DDX3 subfamily members have also been implicated in cellular signaling pathways and are targeted by diverse viruses. In this review, we discuss the considerable body of work on the biochemistry and biology of these proteins, including the recently discovered link of human DDX3 to tumorigenesis.     

DEAD-box helicases as integrators of RNA,nucleotide and protein binding

DEAD-box helicases perform diverse cellular functions in virtually all steps of RNA metabolism from Bacteria to Humans. Although DEAD-box helicases share a highly conserved core domain, the enzymes catalyze a wide range of biochemical reactions. In addition to the well established RNA unwinding and corresponding ATPase activities, DEAD-box helicases promote duplex formation and displace proteins from RNA. They can also function as assembly platforms for larger ribonucleoprotein complexes, and as metabolite sensors. This review aims to provide a perspective on the diverse biochemical features of DEAD-box helicases and connections to structural information. We discuss these data in the context of a model that views the enzymes as integrators of RNA, nucleotide, and protein binding. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

The RNase E of Escherichia coli has at least two binding sites for DEAD-box RNA helicases: functional replacement of RhlB by RhlE

The non-catalytic region of Escherichia coli RNase E contains a protein scaffold that binds to the other components of the RNA degradosome. Alanine scanning yielded a mutation, R730A, that disrupts the interaction between RNase E and the DEAD-box RNA helicase, RhlB. We show that three other DEAD-box helicases, SrmB, RhlE and CsdA also bind to RNase E in vitro. Their binding differs from that of RhlB because it is not affected by the R730A mutation. Furthermore, the deletion of residues 791-843, which does not affect RhlB binding, disrupts the binding of SrmB, RhlE and CsdA. Therefore, RNase E has at least two RNA helicase binding sites. Reconstitution of a complex containing the protein scaffold of RNase E, PNPase and RhlE shows that RhlE can furnish an ATP-dependent activity that facilitates the degradation of structured RNA by PNPase. Thus, RhlE can replace the function of RhlB in vitro. The results in the accompanying article show that CsdA can also replace RhlB in vitro. Thus, RhlB, RhlE and CsdA are interchangeable in in vitro RNA degradation assays.     

The nuclear GUCT domain-containing DEAD-box RNA helicases govern gametophytic and sporophytic development in Physcomitrium patens

Plant Molecular Biology - In Physcomitrium patens, PpRH1/PpRH2 are GUCT-domain-containing DEAD-BOX RNA helicases localize to the nucleus. They are implicated in cell and tissue development in all...     

The newly discovered Q motif of DEAD-box RNA helicases regulates RNA-binding and helicase activity

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.     

Studies on three E. coli DEAD-box helicases point to an unwinding mechanism different from that of model DNA helicases

DEAD-box proteins participate in various aspects of RNA metabolism in all organisms. These RNA-dependent ATPases are usually regarded as double-stranded RNA unwinding enzymes, though in vitro this activity has only been demonstrated for a subset of them. Given their high biological specificity, their equivocal unwinding activity may reflect the noncognate character of the substrates used in vitro. Here, we pinpoint other reasons for this elusiveness. We have compared the ATPase and helicase activities of three E. coli DEAD-box proteins, CsdA, RhlE and SrmB. Whereas the ATPase activity of all proteins is stimulated (albeit to various degree) by long RNAs, only RhlE is stimulated by short oligoribonucleotides. Consistently, all three proteins can unwind RNA duplexes with long single-stranded extensions, but only RhlE is effective when extensions are short or absent. Another critical constraint concerns the length of the duplex region: in the case of RhlE, the ratio (duplex unwound)/(ATP hydrolyzed) drops 1000-fold upon going from 11 to 14 base pairs, indicating a low processivity. Remarkably, allowing for these constraints, all three proteins can unwind substrates with either 5' or 3' extensions (or no extension in the case of RhlE). This behavior, which contrasts with that of well studied SF1 DNA helicases, is discussed in the light of available structural and biochemical data.     

DEAD-box proteins from Escherichia coli exhibit multiple ATP-independent activities

DEAD-box proteins (DBPs) are a widespread class of ATP-dependent RNA helicases that play a key role in unwinding RNA duplexes. In recent years, certain DBPs have also been found to exhibit activities that do not require ATP. To gain a better understanding of prokaryotic RNA metabolism, we investigated whether Escherichia coli DBPs harbor any ATP-independent activities. We show that each of the four E. coli DBPs tested in this study can accelerate the association of cRNA molecules, can stimulate strand displacement, and can function as an RNA chaperone without utilizing ATP. To the best of our knowledge, these prokaryotic DBPs constitute the first examples of proteins that harbor each of these three activities. The identification of these auxiliary functions indicates that the E. coli DBPs are versatile factors that possess significant RNA remodeling activity in addition to their canonical RNA helicase activity and might therefore participate in a greater variety of cellular processes than has been previously appreciated.     

The DEAD-box protein Ded1 unwinds RNA duplexes by a mode distinct from translocating helicases

Helicases unwind RNA or DNA duplexes and displace proteins from nucleic acids in an ATP-dependent fashion. To unwind duplexes, helicases typically load onto one of the two nucleic acid strands, usually at a single-stranded region, and then translocate on this strand in a unidirectional fashion, thereby displacing the complementary DNA or RNA. Here we show that the DEAD-box RNA helicase Ded1 unwinds duplexes in a different manner. Ded1 uses the single-stranded region to gain access to the duplex. Strand separation is directly initiated from the duplex region and no covalent connection between the single strand and the duplex region is required. This new type of helicase activity explains observations with other DEAD-box proteins and may be the prototype for duplex-unwinding reactions in RNA metabolism.     

番茄 DEAD-box RNA 解旋酶基因 SlDEAH1 的克隆及表达分析简

DEAD-box RNA解旋酶是一种特殊的RNA分子伴侣,参与了RNA代谢,包括前体RNA剪接、核糖体合成、RNA降解以及基因表达,并对植物的发育和抗性等也具有重要作用。根据已报道的拟南芥DEAD-box蛋白,通过同源比对,在NCBI据库中筛选得到一个DEAD-box RNA解旋酶同源蛋白,命名为SlDEAH1,并根据其基因序列设计特异引物,应用RT-PCR方法从野生型番茄（Solanum lycopersicum）AC⁺⁺中克隆得到了该基因的全长编码区序列。利用生物学网站、软件及实时荧光定量PCR方法,对其进行生物信息学、表达模式、胁迫及激素处理分析。结果表明：SlDEAH1包括2 073 bp的开放阅读框,编码690个氨基酸残基,其编码蛋白有9个保守结构基序,其所涉及到的ATP结合、ATP水解及RNA结合等功能对于解旋酶活性是至关重要的;表达模式分析表明SlDEAH1基因可能在野生型番茄萼片、叶片发育及果实成熟方面起到重要作用;高温、低温、脱水、伤害、盐胁迫不同程度的诱导了SlDEAH1的表达,但在根中该基因的表达受盐胁迫抑制;ABA、ACC、IAA、GA₃、MeJA和ZT均不同程度诱导了SlDEAH1的表达,其中ABA诱导效应最为明显。这些结果为进一步研究SlDEAH1在番茄发育和胁迫响应中的功能奠定了基础。     

The DEAD-box RNA helicase SrmB is involved in the assembly of 50S ribosomal subunits in Escherichia coli

Ribosome 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.     

Escherichia coli DNA helicases: mechanisms of DNA unwinding

DNA helicases are ubiquitous enzymes that catalyse the unwinding of duplex DNA during replication, recombination and repair. These enzymes have been studied extensively; however, the specific details of how any helicase unwinds duplex DNA are unknown. Although it is clear that not all helicases unwind duplex DNA in an identical way, many helicases possess similar properties, which are thus likely to be of general importance to their mechanism of action. For example, since helicases appear generally to be oligomeric enzymes, the hypothesis is presented in this review that the functionally active forms of DNA helicases are oligomeric. The oligomeric nature of helicases provides them with multiple DNA-binding sites, allowing the transient formation of ternary structures, such that at an unwinding fork, the helicase can bind either single-stranded and duplex DNA simultaneously or two strands of single-stranded DNA. Modulation of the relative affinities of these binding sites for single-stranded versus duplex DNA through ATP binding and hydrolysis would then provide the basis for a cycling mechanism for processive unwinding of DNA by helicases. The properties of the Escherichia coli DNA helicases are reviewed and possible mechanisms by which helicases might unwind duplex DNA are discussed in view of their oligomeric structures, with emphasis on the E. coli Rep, RecBCD and phage T7 gene 4 helicases.     

RNA helicases in bacteria

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Identification of the sites of action of SrmB, a DEAD-box RNA helicase involved in Escherichia coli ribosome assembly

DEAD-box RNA-dependent ATPases are ubiquitous enzymes that participate in nearly all processes involving RNA, but their detailed molecular functions remain generally unknown. SrmB, one of the five Escherichia coli DEAD-box proteins, participates in the assembly of the large ribosomal subunit notably by facilitating the incorporation of L13, one of the ribosomal proteins that bind 23S rRNA earliest. Previously, we showed that SrmB is tethered to nascent ribosome through interactions with L4, L24 and the region from domain I of 23S rRNA that binds them. To identify the sites of action of SrmB, we have characterized rRNA mutations that bypass SrmB requirement. Five of them affect the same position from two repeated heptanucleotides in domain II of 23S rRNA, whereas two others affect a complementary hexanucleotide in 5S rRNA. Thus the sites of action of SrmB differ from its tethering site. In the mature ribosome, one of the heptanucleotides participates in a highly compact structure that contacts L13, the '1024 G-ribo wrench'. In addition, we have observed that the assembly defect of ΔsrmB cells worsens as rRNA synthesis increases. Based on these results, we propose two non-exclusive scenarios for the role of SrmB in ribosome assembly.