DEAH helicases participate in pre-messenger RNA splicing and ribosome biogenesis. The structure of yeast Prp43p-ADP reveals the homology of DEAH helicases to DNA helicases and the presence of an oligonucleotide-binding motif. A b-hairpin from the second RecA domain is wedged between two carboxyterminal domains and blocks access to the occluded RNA binding site formed by the RecA domains and a C-terminal domain. ATP binding and hydrolysis are likely to induce conformational changes in the hairpin that are important for RNA unwinding or ribonucleoprotein remodelling. The structure of Prp43p provides the framework for functional and genetic analysis of all DEAH helicases.
eIF4A is a key component in eukaryotic translation initiation; however, it has not been clear how auxiliary factors like eIF4B and eIF4G stimulate eIF4A and how this contributes to the initiation process. Based on results from isothermal titration calorimetry, we propose a two-site model for eIF4A binding to an 83.5 kDa eIF4G fragment (eIF4G-MC), with a high- and a low-affinity site, having binding constants KD of ∼50 and ∼1000 nM, respectively. Small angle X-ray scattering analysis shows that the eIF4G-MC fragment adopts an elongated, well-defined structure with a maximum dimension of 220 Å, able to span the width of the 40S ribosomal subunit. We establish a stable eIF4A–eIF4B complex requiring RNA, nucleotide and the eIF4G-MC fragment, using an in vitro RNA pull-down assay. The eIF4G-MC fragment does not stably associate with the eIF4A–eIF4B–RNA-nucleotide complex but acts catalytically in its formation. Furthermore, we demonstrate that eIF4B and eIF4G-MC act synergistically in stimulating the ATPase activity of eIF4A.
Three families of nucleic acid-dependent ATPases (DEAH/RHA, Ski2-like, and NS3/NPH-II), termed the DExH ATPases, are thought to execute myriad functions by processive, ATP-dependent, 3' to 5' translocation along single-stranded nucleic acid. While the mechanism of translocation of the viral NS3/NPH-II family has been studied extensively, it has not been clear if or how the principles that have emerged for this family extend to the other two families. Here we report the crystal structure of the yeast DEAH/RHA family ATPase Prp43p, which functions in splicing and ribosome biogenesis, in complex with poly-uracil and a nonhydrolyzable ATP analog. The structure reveals a conserved DEAH/RHA-specific variation of motif Ib within the RecA1 domain of the catalytic core, in which the motif elongates as a β-hairpin that bookends the 3' end of a central RNA stack, a function that in the viral and Ski-2 families is performed by an auxiliary domain. Supporting a fundamental role in translocation, mutations in this hairpin abolished helicase activity without affecting RNA binding or ATPase activity. While the structure reveals differences with viral ATPases in the RecA1 domain, our structure demonstrates striking similarities with viral ATPases in the RecA2 domain of the catalytic core, including both a prominent β-hairpin that bookends the 5' end of the RNA stack and a dynamic motif Va that is implicated in mediating translocation. Our crystal structure, genetic, and biochemical experiments, as well as comparisons with other DExH ATPases, support a generalized mechanism for the DExH class of helicases involving a pair of bookends that inchworm along RNA.
The sensitive to lysis D (SlyD) protein from Escherichia coli is related to the FK506‐binding protein family, and it harbours both peptidyl‐prolyl cis–trans isomerase (PPIase) and chaperone‐like activity, preventing aggregation and promoting the correct folding of other proteins. Whereas a functional role of SlyD as a protein‐folding catalyst in vivo remains unclear, SlyD has been shown to be an essential component for [Ni–Fe]‐hydrogenase metallocentre assembly in bacteria. Interestingly, the isomerase activity of SlyD is uniquely modulated by nickel ions, which possibly regulate its functions in response to external stimuli. In this work, we investigated the solution structure of SlyD and its interaction with nickel ions, enabling us to gain insights into the molecular mechanism of this regulation. We have revealed that the PPIase module of SlyD contains an additional C‐terminal α‐helix packed against the catalytic site of the domain; unexpectedly, our results show that the interaction of SlyD with nickel ions entails participation of the novel structural features of the PPIase domain, eliciting structural alterations of the catalytic pocket. We suggest that such conformational rearrangements upon metal binding underlie the ability of nickel ions to regulate the isomerase activity of SlyD.
Helicases are ubiquitous enzymes that participate in every aspect of nucleic acid metabolism. The DEAH/RHA family of helicases are involved in a variety of cellular processes including transcriptional and translational regulation, pre-mRNA splicing, pre-rRNA processing, mRNA export and decay, in addition to the innate immune response. Recently, the first crystal structures of a DEAH/RHA helicase unveiled the unique structural features of this helicase family. These structures furthermore illuminate the molecular mechanism of these proteins and provide a framework for analysis of their interaction with nucleic acids, regulatory proteins and large macromolecular complexes.
INTRODUCTION: "Improving the employment rate of college students" directly affects the stability of the country and society and the healthy development of the industry market. The traditional graduate employment rate model only predicts the future employment rate based on changes in historical employment data in previous years. OBJECTIVES: Quantify the employment factors and solve the employment problems in colleges and universities in a targeted manner. METHODS: We construct a credible employment prediction model for college graduates based on LightGBM. RESULTS: We use the model to predict the employment status of students and obtain the special importance which is important to employment of college students. CONCLUSION: The final result shows that our Model performs well in the two indicators of accuracy and model quality.
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