Structure of the DEAH/RHA ATPase Prp43p bound to RNA implicates a pair of hairpins and motif Va in translocation along RNA.

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 ß-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 ß-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.

Structural analysis of RNA helicases with small-angle X-ray scattering.

Small-angle X-ray scattering (SAXS) is a structural characterization method applicable to biological macromolecules in solution. The great advantage of solution scattering is that the systems can be investigated in near-physiological conditions and their response to external changes can also be easily investigated. In this chapter, we discuss the application of SAXS for studying the conformation of helicases alone and in complex with other biological macromolecules. The DEAD-box helicase eIF4A and the DEAH/RHA helicase Prp43 are investigated for their solution structures, and the analysis of the collected scattering data is presented. A wide range of methods for analysis of SAXS data are presented and discussed. Ab initio methods can be used to yield low-resolution solution structures, and when models with atomic resolution are available, these can be included to aid the determination of solution structures. Using such prior information relating to the systems studied and applying a variety of methods, substantial insight can be gained about solution structures and interactions of biological macromolecules through small-angle scattering.

The function and architecture of DEAH/RHA helicases.

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.

Synergistic activation of eIF4A by eIF4B and eIF4G.

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.

Structural basis for the function of DEAH helicases.

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 beta-hairpin from the second RecA domain is wedged between two carboxy-terminal 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.

The interaction of the Escherichia coli protein SlyD with nickel ions illuminates the mechanism of regulation of its peptidyl-prolyl isomerase activity.

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 alpha-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.

[Effect of different acupuncture needle-retaining time on hemorheology in ischemic stroke patients].

OBJECTIVE: To observe the effect of different needle-retaining duration on hemorheology in ischemic stroke patients in order to explore its underlying mechanism in the treatment of ischemic stroke. METHODS: A total of 251 ischemic apoplexy patients were, according to their state (mild, moderate and severe) of disease, randomly divided into 20 min, 40 min and 60 min groups respectively. Acupoints Jianyu (LI15), Quchi (LI11), Waiguan (TE5), Hegu (LI4), Futu (ST32), Zusanli (ST 36), etc. were punctured and stimulated electrically with parameters of 2 Hz, 2-6 mA for 20 min, 40 min and 60 min separately, once daily for 10 times. In addition, the patients were also treated with medicines for lowering intracranial pressure as manicol, etc. Higher shearing rate (HSR) and lower shearing rate (LSR) of whole blood viscosity (etab), plasma viscosity (etap), hematocrit (HCT) and profibrin were detected separately before and after the treatment. RESULTS: After acupuncture treatment, of the 83, 80 and 82 cases in 20 min, 40 min and 60 min groups, 3 (3.61%), 4 (5.00%) and 9 (10.98%) were cured basically, 15 (18.07%), 17 (21.25%) and 21 (25.61%) experienced remarkable improvement in their symptoms and signs, 24 (28.92%), 33 (41.25%) and 40 (48.78%) had an improvement, 41 (49.40%), 26 (32.50%) and 12 (14.63%) failed in the treatment, with the markedly effective rates being 21.68% , 26.25% and 36.59% and the effective rates being 50.60%, 67.50% and 85.37% separately. The effective rate of 60 min group was significantly higher than those of 20 min and 40 min groups (P<0.01, 0.05). Self-comparison showed that HSR, LSR of etab, etap, HCT and profibrin levels decreased considerably in comparison with pre-treatment in each group (P<0.01). Difference values of HSR, LSR, etap, HCT and profibrin (pre-treatment--those of post-treatment) in 60 min group were significantly higher than those of 20 min and 40 min groups (P<0.05, 0.01), and those of HSR, HCT and profibrin of 40 min group were markedly higher than those of 20 min group (P<0.01, 0.05). The therapeutic effect of 60 min group was the best. CONCLUSION: Acupuncture can effectively improve stroke patients' symptoms and signs, and lower various parameters of hemorheology.

An observation on the acupuncture treatment of ischemic wind-stroke with different lengths of needle-retaining time.

OBJECTIVE: To observe the effects of different lengths of needle-retaining time on the therapeutic results of acupuncture treatment for ischemic wind-stroke. METHODS: According to the severity of disease, 251 cases of ischemic wind-stroke were randomly divided into 3 groups and treated by acupuncture, with the needle-retaining time lasting 20, 40, and 60 minutes respectively. Observation was mainly focused on the scores of nervous dysfunction and the clinical therapeutic results. RESULTS: The comparison of the scores of nervous dysfunction in the 3 groups before and after the treatment showed that under Wilcoxon's pair-match symbol rank test, except for horizontal gaze, other indexes all showed significant differences (P < 0.01). In the between-group comparison before and after the treatment, there showed significant or very significant differences in the indexes of myodynamia of the upper limb, myodynamia of hand and the total scores (P < 0.05, P < 0.01). In the between-group comparison by Mann-Whitney rank test of the indexes of myodynamia of upper limb, myodynamia of hand and the total scores, the 20-min. group and the 60-min. group showed significant differences (P < 0.05); and 40-min. group and 60-min. group showed significant differences in myodynamia of upper limb (P < 0.05). In comparison of the therapeutic effects of the 3 groups by Kruskal-wallis rank test, there showed very significant differences (P < 0.01). In the between-group comparison by Mann-Whitney rank test, the 40-min.group and 60-min. group showed significant differences (P = 0.013); while the 20-min. group and 60-min. group showed very significant differences (P = 0.000). CONCLUSION: There is a time-effect relationship between the length of needle-retaining time and the therapeutic effects of acupuncture for ischemic wind-stroke.

[Effects of acupuncture on the cortical functional areas activated by index finger motion in the patient with ischemic stroke].

OBJECTIVE: To observe the effect of acupuncture on cortical functional areas of the patient with ischemic stroke activated by the index finger motion. METHODS: The cortical magnetic resonance imaging (fMRI) were carried out in 15 cases of ischemic stroke during the index finger motion at acupuncture or non-acupuncture. The distribution of the cortical functional areas activated and the size of the activated region and the intension of signals were measured. RESULTS: The finger motion with no acupuncture could activate the contralateral primary somatomotor area (M1), contralateral premotor area (PMA) and contralateral first somatosensory area (S1). The finger motion with acupuncture could activate the same areas and also activate ipsilateral M1, focus area contralateral superior parietal lobule, contralateral superior temporal gyrus, and contralateral insular lobe, etc.. Both the area of the activated region and the minimum signal in the finger motion with acupuncture were statistically significantly larger than those in finger motion with no acupuncture. CONCLUSION: Rehabilitation of motor functions of the patient with ischemic stroke by acupuncture is related with improvement of blood circulation functional area in the cortex.