Gene name - maleless
Synonyms - male-lethal
Cytological map position - 42A1--42A4
Function - RNA/DNA helicase
Symbol - mle
FlyBase ID: FBgn0002774
Genetic map position - 2-55.2
Classification - DEAH-box subfamily ATP-dependent helicase
Cellular location - nuclear
|Recent literature||Prabu, J. R., Muller, M., Thomae, A. W., Schussler, S., Bonneau, F., Becker, P. B. and Conti, E. (2015). Structure of the RNA helicase MLE reveals the molecular mechanisms for uridine specificity and RNA-ATP coupling. Mol Cell 60: 487-499. PubMed ID: 26545078
The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. This study identified a stable MLE core comprising the DExH helicase module and two auxiliary domains: a dsRBD and an OB-like fold. MLEcore is an unusual DExH helicase that can unwind blunt-ended RNA duplexes and has specificity for uridine nucleotides.The 2.1 A resolution structure of MLEcore was determined bound to a U10 RNA and ADP-AlF4. The OB-like and dsRBD folds bind the DExH module and contribute to form the entrance of the helicase channel. Four uridine nucleotides engage in base-specific interactions, rationalizing the conservation of uridine-rich sequences in critical roX substrates. roX2 binding is orchestrated by MLE's auxiliary domains, which is prerequisite for MLE localization to the male X chromosome. The structure visualizes a transition-state mimic of the reaction and suggests how eukaryotic DEAH/RHA helicases couple ATP hydrolysis to RNA translocation.
|Cugusi, S., Li, Y., Jin, P. and Lucchesi, J.C. (2016). The Drosophila helicase MLE targets hairpin structures in genomic transcripts. PLoS Genet 12: e1005761. PubMed ID: 26752049
RNA hairpins are a common type of secondary structure that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi) and microRNA (miRNA) pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss) RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE has been shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. This study reports that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, it was shown that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. The study uses two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway.
|Ilik, I. A., Maticzka, D., Georgiev, P., Gutierrez, N. M., Backofen, R. and Akhtar, A. (2017). A mutually exclusive stem-loop arrangement in roX2 RNA is essential for X-chromosome regulation in Drosophila. Genes Dev 31(19): 1973-1987. PubMed ID: 29066499
The X chromosome provides an ideal model system to study the contribution of RNA-protein interactions in epigenetic regulation. In male flies, roX long noncoding RNAs (lncRNAs) harbor several redundant domains to interact with the ubiquitin ligase male-specific lethal 2 (MSL2) and the RNA helicase Maleless (MLE) for X-chromosomal regulation. However, how these interactions provide the mechanics of spreading remains unknown. By using the uvCLAP (UV cross-linking and affinity purification) methodology, which provides unprecedented information about RNA secondary structures in vivo, the minimal functional unit of roX2 RNA was identified. By using wild-type and various MLE mutant derivatives, including a catalytically inactive MLE derivative, MLE(GET), this study showed that the minimal roX RNA contains two mutually exclusive stem-loops that exist in a peculiar structural arrangement: When one stem-loop is unwound by MLE, an alternate structure can form, likely trapping MLE in this perpetually structured region. This functional unit is necessary for dosage compensation, as mutations that disrupt this formation lead to male lethality. Thus, it is proposed that roX2 lncRNA contains an MLE-dependent affinity switch to enable reversible interactions of the MSL complex to allow dosage compensation of the X chromosome.
|Lv, M., Yao, Y., Li, F., Xu, L., Yang, L., Gong, Q., Xu, Y. Z., Shi, Y., Fan, Y. J. and Tang, Y. (2019). Structural insights reveal the specific recognition of roX RNA by the dsRNA-binding domains of the RNA helicase MLE and its indispensable role in dosage compensation in Drosophila. Nucleic Acids Res. PubMed ID: 30649456
In Drosophila, dosage compensation globally upregulates the expression of genes located on male single X-chromosome. Maleless (MLE) helicase plays an essential role to incorporate the roX lncRNA into the dosage compensation complex (MSL-DCC), and such function is essentially dependent on its dsRNA-binding domains (dsRBDs). This study reports a 2.90A crystal structure of tandem dsRBDs of MLE in complex with a 55mer stem-loop of roX2 (R2H1). MLE dsRBDs bind to R2H1 cooperatively and interact with two successive minor grooves and a major groove of R2H1, respectively. The recognition of R2H1 by MLE dsRBDs involves both shape- and sequence-specificity. Moreover, dsRBD2 displays a stronger RNA affinity than dsRBD1, and mutations of key residues in either MLE dsRBD remarkably reduce their affinities for roX2 both in vitro and in vivo. In Drosophila, the structure-based mle mutations generated using the CRISPR/Cas9 system, are partially male-lethal and indicate the inter-regulation among the components of the MSL-DCC at multiple levels. Hence, this research provides structural insights into the interactions between MLE dsRBDs and R2H1 and facilitates a deeper understanding of the mechanism by which MLE tandem dsRBDs play an indispensable role in specific recognition of roX and the assembly of the MSL-DCC in Drosophila dosage compensation.
|Ankush Jagtap, P. K., Muller, M., Masiewicz, P., von Bulow, S., Hollmann, N. M., Chen, P. C., Simon, B., Thomae, A. W., Becker, P. B. and Hennig, J. (2019). Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless. Nucleic Acids Res. PubMed ID: 30805612
Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. This study determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. The NMR and SAXS data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAXK RNA binding motifs. NMR titrations combined with filter binding experiments and isothermal titration calorimetry (ITC) document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.
|Lv, M., Yao, Y., Li, F., Xu, L., Yang, L., Gong, Q., Xu, Y. Z., Shi, Y., Fan, Y. J. and Tang, Y. (2019). Structural insights reveal the specific recognition of roX RNA by the dsRNA-binding domains of the RNA helicase MLE and its indispensable role in dosage compensation in Drosophila. Nucleic Acids Res 47(6): 3142-3157. PubMed ID: 30649456
In Drosophila, dosage compensation globally upregulates the expression of genes located on male single X-chromosome. Maleless (MLE) helicase plays an essential role to incorporate the roX lncRNA into the dosage compensation complex (MSL-DCC), and such function is essentially dependent on its dsRNA-binding domains (dsRBDs). This paper reports a 2.90A crystal structure of tandem dsRBDs of MLE in complex with a 55mer stem-loop of roX2 (R2H1). MLE dsRBDs bind to R2H1 cooperatively and interact with two successive minor grooves and a major groove of R2H1, respectively. The recognition of R2H1 by MLE dsRBDs involves both shape- and sequence-specificity. Moreover, dsRBD2 displays a stronger RNA affinity than dsRBD1, and mutations of key residues in either MLE dsRBD remarkably reduce their affinities for roX2 both in vitro and in vivo. In Drosophila, the structure-based mle mutations generated using the CRISPR/Cas9 system, are partially male-lethal and indicate the inter-regulation among the components of the MSL-DCC at multiple levels. Hence, this research provides structural insights into the interactions between MLE dsRBDs and R2H1 and facilitates a deeper understanding of the mechanism by which MLE tandem dsRBDs play an indispensable role in specific recognition of roX and the assembly of the MSL-DCC in Drosophila dosage compensation.
Dosage compensation (compensation for having different numbers of X chromosomes) is a crucial developmental process: without it, females would have twice the amount of X-linked gene product as males, because females have two X chromosomes to a single X chromosome for males. In Drosophila equalization of the amounts of gene product produced by X-linked genes in the two sexes is achieved by hypertranscription of the single male X chromosome. This process is controlled by a set of male-specific lethal (msl) genes that appear to act at the level of chromatin structure. Four proteins (encoded by the male-specific lethal genes) are required for dosage compensation; they associate with the X chromosome in males but not in females. The sequences of the MSL proteins contain several motifs potentially associated with transcriptional control.
Clues to an understanding of the role of Mle in dosage compensation come from three sources: biochemical analysis, genetic analysis, and information about physical interaction of Mle with polytene chromosomes. Mle protein has been shown to possess RNA/DNA helicase activity, adenosine triphosphatase (ATPase) activity and single-stranded (ss) RNA/ssDNA binding activities. Helicases are DNA and RNA unwinding proteins. The helicase activity demonstrates a degree of substrate specificity. Mle displaces substrates containing single stranded RNA regions more efficiently than substrates containing single stranded DNA regions. A mutant of mle (mle-GET) was created that contains a glutamic acid in place of lysine in the conserved ATP binding site A. In vitro biochemical analysis shows that this mutation abolishes both ATPase and helicase activities of MLE but affects the ability of MLE to bind to single stranded DNA and RNA less severly. In vivo, Mle-GET protein can still localize to the male X chromosome but fails to complement mle1 mutant males. These results indicate that the ATPase/helicase activities are essential functions of Mle for dosage compensation but that the interaction of Mle with chromosomes is largely independent of the helicase activity. In other words, the role of Mle in dosage compensation requires at least two functions: (1) the binding to chromosomes, and (2) a helicase activity (Lee, 1997).
How does Mle protein bind to chromosomes? Association of MLE with the male X chromosome is ribonuclease sensitive. Overexpression of Mle or its carboxyl terminus (which includes glycine-rich repeats) reveals an RNase-sensitive affinity for all chromosome arms. These results suggest that nascent messenger RNA transcripts or a hypothetical RNA component of chromatin plays a critical role in the biochemical mechanism of dosage compensation (Richter, 1996).
Part of the Mle protein structure is a conserved RNA binding motif. One RNA binding domain, termed the double-stranded RNA-binding domain (dsRBD), was first identified as 70 residue repeat motifs in three proteins: dsRNA-dependent (DAI) protein kinase, Xenopus RNA-binding protein xlrbpa and Drosophila maternal effect protein Staufen. Two copies of the domain have been detected in helicases: in mammalian RNA helicase A and in Drosophila Maleless. The helicase domain appears to be insufficient on its own to promote helicase activity and additional RNA-binding capacity must be supplied either as domains adjacent to the helicase domain, as in Mle, or by bound RNA recognizing partners (Gibson, 1994).
What is the hierarchy of gene and protein function in dosage compensation? At the top of the hierarchy is Sex lethal, the master regulator of sex determination in Drosophila. Sex lethal regulates the splicing of Msl-2 producing a functional Msl-2 protein in males only (Kelley, 1995 and 1997). The other three male specific lethals are not differentially spliced, although putative SXL-binding sites are also found in the 3' UTR of a subset of MSL-1 transcripts. Msl-2, spliced into a functional form in males, serves to heighten transcriptional activation of the solitary X chromosome. MSL-2 acts in concert with three partners in this task: Maleless, Male-specific lethal-1 and Male specific lethal-3. It is thought that Msl-2 acts in some manner to promote the binding of the other three factors to chromatin. However, Mle might not interact specifically with chromatin but rather with RNA. Is chromosomal RNA the direct target of Mle in dosage compensation, or does RNA have an unknown role in assembling the male specific lethal gene activation complex to the chromosome? There is no reason to assume these possible roles for Mle are mutually exclusive, and both may be involved.
Chromatin is thought to be the target of the MSL proteins. A specific acetylated isoform of Histone H4, H4Ac16, is also detected predominantly on the male X chromosome. Mle and Msl-1 bind to the X chromosome in an identical pattern and the pattern of H4Ac16 on the X chromosome is largely coincident with that of Mle/Msl-1. H4Ac16 was not detected on the X chromosome in homozygous male specific lethal mutant males, correlating with the lack of dosage compensation in these mutants. These data suggest that synthesis or localization of H4Ac16 is controlled by the dosage compensation regulatory hierarchy. Dosage compensation may involve H4Ac16 function, potentially through interaction with the product of the msl genes (Bone, 1994).
The role of Maleless in the chromosomal activation hierarchy remains unclear. Its DNA helicase activity may contribute to destabilizing chromatin structure, analgous to ATP-dependent nucleosomal disruption by the SWI/SNF and Drosophila NURF complexes (see Imitation SWI and Brahma). Its RNA helicase activity could facilitate transcription by altering the structure of nascent RNA, a process that can stimulate reinitiation and/or elongation. Alternatively, Mle may interact with a hypothetical structural RNA component of the chromosome. There is precedence for such RNA in the existence of X chromosome associated RNA on the X. Interestingly, while Mle is released following RNase treatment, the other male specific lethal proteins remain associated with the chromosome (Richter, 1996). This suggests that the several distinct known biochemical functions of male specific lethal proteins will lend themselves to distinct independent functions in dosage compensation (Lee, 1997).
The ribonucleoprotein Male Specific Lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila males. Beginning at 3 h of development the MSL complex binds transcribed X-linked genes and modifies chromatin. A subset of MSL complex proteins, including MSL1 and MSL3, is also necessary for full expression of autosomal heterochromatic genes in males, but not females. Loss of the non-coding roX RNAs, essential components of the MSL complex, lowers the expression of heterochromatic genes and suppresses position effect variegation (PEV) only in males, revealing a sex-limited disruption of heterochromatin. MLE, but not Jil-1 kinase, was found to contribute to heterochromatic gene expression. To determine if identical regions of roX RNA are required for dosage compensation and heterochromatic silencing, a panel of roX1 transgenes and deletions was tested; the X chromosome and heterochromatin functions were found to be separable by some mutations. Widespread autosomal binding of MSL3 occurs before and after localization of the MSL complex to the X chromosome at 3 h AEL. Autosomal MSL3 binding was dependent on MSL1, supporting the idea that a subset of MSL proteins associates with chromatin throughout the genome during early development. It is postulated that this binding may contribute to the sex-specific differences in heterochromatin that have been noted (Koya, 2015).
A central question raised by this study is how factors known for their role in X chromosome dosage compensation also modulate autosomal heterochromatin. Although the MSL proteins were first identified by their role in X chromosome compensation, homologues of these proteins participate in chromatin organization, DNA repair, gene expression, cell metabolism and neural function throughout the eukaryotes. Furthermore, flies contain a distinct complex, the Non-Sex specific Lethal (NSL) complex, containing MOF and the MSL orthologs NSL1, NSL2 and NSL3. The essential NSL complex is broadly associated with promoters throughout the fly genome, where it acetylates multiple H4 residues. In light of the discovery that the MSL proteins represent an ancient lineage of chromatin regulators, it is unsurprising that members of this complex fulfill additional functions (Koya, 2015).
An alternative hypothesis for the dosage compensation of male X-linked genes proposes that the MSL proteins are general transcription regulators, and recruitment of these factors to the male X chromosome reduces autosomal gene expression, thus equalizing the X:A expression ratio. Arguing against this idea are ChIP studies finding that the MSL complex, and engaged RNA polymerase II, are increased within the bodies of compensated X-linked genes. In agreement with this, a study that normalized expression to genomic DNA concluded that compensation increases the expression of male X-linked genes. The current study now reveals that autosomal heterochromatic genes are indeed dependent on a subset of MSL proteins for full expression. However, native heterochromatic genes make up only 4% of autosomal genes, and their misregulation is not expected to compromise genome-wide expression studies normalized to autosomal expression (Koya, 2015).
Expression of heterochromatic genes is thought to involve mechanisms to overcome the repressive chromatin environment. It is possible that a complex composed of roX RNA and a subset of MSL proteins participates in this process. This would explain why heterochromatic genes are particularly sensitive to the loss of these factors. Alternatively, it is possible that roX and MSL proteins participate in heterochromatin assembly. This would explain the simultaneous disruption of heterochromatic gene expression and suppression of PEV at transgene insertions (Koya, 2015).
Heterochromatin assembly is first detected at 3-4 h AEL, a time when MSL3 is bound throughout the genome. Intriguingly, studies from yeast identify a role for H3K4 and H4K16 acetylation in formation of heterochromatin. Active deacetylation of H4K16ac is necessary for spreading of chromatin-based silencing in yeast, demonstrating the need for a sequential and ordered series of histone modifications (Koya, 2015).
As MOF is responsible for the majority of H4K16ac in the fly, a MOF-containing complex could fulfill a similar role during heterochromatin formation. While this study found a significant effect of MOF in expression only on the X and 4th chromosomes, it is possible that examination of a larger number of genes would reveal a more widespread autosomal effect (Koya, 2015).
In roX1 roX2 males the 4th chromosome displays stronger suppression of PEV and more profound gene misregulation than do other heterochromatic regions. This is consistent with the observation that heterochromatin on the 4th chromosome is genetically and biochemically different from that on other chromosomes. Loss of roX RNA leads to misregulation of genes in distinct genomic regions, the dosage compensated X chromosome and autosomal heterochromatin. This study found that the regulation of these two groups is, to some extent, genetically separable. MSL2, which binds roX1 RNA and is an essential member of the dosage compensation complex, is not required for full expression of heterochromatic genes in males. Ectopic expression of MSL2 in females induces formation of MSL complexes that localize to both X chromosomes, inducing inappropriate dosage compensation. As would be expected from the lack of a role for MSL2 in autosomal heterochromatin in males, ectopic expression of this protein in females has no effect on PEV (Koya, 2015).
Elegant, high-resolution studies reveal that MLE and MSL2 bind essentially indistinguishable regions of roX1. Three prominent regions of MLE/MSL2 binding have been identified, one overlapping the 3' stem loop. This stem loop incorporates a short 'roX box' consensus sequence that is present in D. melanogaster roX1 and roX2, and conserved in roX RNAs in related species (Koya, 2015).
An experimentally supported explanation for the concurrence of MLE and MSL2 binding at the 3' stem loop is that MLE, an ATP-dependent RNA/DNA helicase, remodels this structure to permit MSL2 binding. The finding that disruption of this stem blocks dosage compensation but does not influence heterochromatic integrity is consistent with participation of roX1 in two processes that differ in MSL2 involvement. However, a region surrounding the stem loop is required for the heterochromatic function of roX1, as roX1Δ10, removing the stem loop and upstream regions, is deficient in both dosage compensation and heterochromatic silencing. Further differentiating these processes is the finding that low levels of roX RNA from a repressed transgene fully rescue heterochromatic silencing, but not dosage compensation. An intriguing question raised by this study is why the sexes display differences in autosomal heterochromatin (Koya, 2015).
The chromatin content of males and females are substantially different as XY males have a single X and a large, heterochromatic Y chromosome. It is speculated that this has driven changes in how heterochromatin is established or maintained in one sex. A search for the genetic regulators of the sex difference in autosomal heterochromatin eliminated the Y chromosome and the conventional sex determination pathway, suggesting that the number of X chromosomes determines the sensitivity of autosomal heterochromatin to loss of roX activity. Interestingly, the amount of pericentromeric X heterochromatin, rather than the euchromatic 'numerator' elements, appears to be the critical factor. The recognition that heterochromatin displays differences in the sexes, and that a specific set of proteins are required for normal function of autosomal heterochromatin in males suggests a useful paradigm for the evolution of chromatin in response to genomic content (Koya, 2015).
Exons - 5
Bases in 3' UTR - 450
Maleless contains seven short segments that define a superfamily of known and putative RNA and DNA helicases. This group includes proteins from E. coli, yeast, Drosophila, mammals, and DNA and RNA viruses. Several have been shown to have DNA or RNA helicase activity, or nucleic acid-dependent ATPase activity. The members of the superfamily share seven distinct conserved segments. Two segments contain the "A" and "B" sites of an NTP binding domain motif; no activity has been assigned individually to the others. Mle contains similarity to each of the conserved segments, including the six amino acids that are invariant throughout the superfamily. The location of similarity within the Mle protein falls in the central portion and spans approximately 360 amino acids, from amino acids 400 to 760 (Kuroda, 1991).
Within the superfamily, several subfamilies of related proteins can be distinguished by two criteria: particular signature motifs [e.g., the sequence DEAD (Asp, Glu, Ala, Asp) in segment II] and a higher overall sequence identity throughout the entire helicase domain. On the basis of these criteria, Mle is a member of a subfamily of putative helicase proteins, the DEAH family. The first described members of this family are three proteins involved in pre-mRNA processing in yeast (PRP2, PRP16 and PRP22). The similarity between Mle and PRP2, PRP16 and PRP22 is extensive throughout the putative helicase domain. Similarity between Mle and DEAH family members extends approximately 100 amino acids beyond the last conserved segment (VI) of the large superfamily. Despite the high overall similarity of Mle to DEAH family members, Mle carries a difference in the proposed signature sequence, with the sequence DEIH rather than DEAH in segment II of the putative ATP-binding motif. Outside of the putative helicase domain, Mle does not show significant sequence similarity to proteins in current data bases. The C-terminal region contains nine imperfect repeats of a glycine-rich sequence. The posterior embryonic determinant Vasa, a member of the superfamily of putative helicases, encodes six copies of a glycine-rich heptad repeat (Kuroda, 1991 and references).
The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. This study identified a stable MLE core comprising the DExH helicase module and two auxiliary domains: a dsRBD and an OB-like fold. MLEcore is an unusual DExH helicase that can unwind blunt-ended RNA duplexes and has specificity for uridine nucleotides. The 2.1 Å resolution structure of MLEcore bound to a U10 RNA and ADP-AlF4 was determined. The OB-like and dsRBD folds bind the DExH module and contribute to form the entrance of the helicase channel. Four uridine nucleotides engage in base-specific interactions, rationalizing the conservation of uridine-rich sequences in critical roX substrates. roX2 binding is orchestrated by MLE's auxiliary domains, which is prerequisite for MLE localization to the male X chromosome. The structure visualizes a transition-state mimic of the reaction and suggests how eukaryotic DEAH/RHA helicases couple ATP hydrolysis to RNA translocation (Prabu, 2015).
It is concluded that the roX RNAs are a paradigm for how lncRNAs function to assemble ribonucleoprotein complexes. The results provide a structural underpinning to the observation of interdependence between MLE and roX RNA for X chromosome targeting and lay the foundation for a future mechanistic analysis of roX RNA remodeling by MLE. It is envisioned that MLE may load on the helical regions of the roX RNAs in an ATP-independent manner via the exposed surface of dsRBD2 and then thread the 3' end of the RNA into the helicase core, rationalizing why the roX boxes are found near the 3' end of the roX RNAs. The finding that MLE also binds uridine-rich sequences at the 5' end of the roX RNAs might be due to the accumulation of the helicase as it progresses from the 3' to the 5' end of the transcript or due to direct loading by a partial opening of the DExH ring. In addition to the canonical helicase interactions with the sugar-phosphate backbone of the RNA, the OB-like fold of MLE can recognize uridine-rich sequences by base-specific interactions. The pattern of uridine-specific interactions observed in the structure (UxUUU) does not exactly match the sequence motif of the roX boxes (UUUU). Imperfectly matching pockets might tune RNA binding strength and the efficiency of translocation, which are inversely correlated. Along these lines, slowed unwinding as MLE interacts with the uridine-rich sequences could provide a window of opportunity for recruiting MSL proteins near the base of helicase, where the 3' end of the unwound strand of the roX RNA emerges, thus contributing to subsequent steps of complex assembly (Prabu, 2015).
Besides revealing the roles of the auxiliary domains in imparting specific uridine-binding and dsRNA-unwinding properties to MLE, the structure reported in this study provides the snapshot of a eukaryotic DExH helicase in the transient 'on' state, showing how the nucleotide and RNA ligands are coupled. The structure of the Ski2-like protein Hel308 showed that unwinding is achieved by a β-hairpin in the RecA2 domain and suggested that translocation is achieved by a ratchet helix in the HA2 domain. MLE operates with different mechanisms, because it has different auxiliary domains and different features that line the unwinding entrance of the helicase channel. The interpretation from the structural data on MLE is that global and local changes in RecA2 propel the movement in the 3'-5' direction, with a loop jointed on RecA2 acting as the pawl in the ratcheting mechanism. It is proposed that a conceptually similar mechanism for 3'-5' directional translocation might be at play in other eukaryotic RHA/DEAH helicases (Prabu, 2015).
date revised: 15 July 97
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