Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly

Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3'-5' directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3' end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a 'splayed' conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.     

Pivotal roles for the receiver domain in the mechanism of action of the response regulator RamR of Streptomyces coelicolor

The response regulator RamR activates expression of the ramCSAB operon, the source of the morphogenetic peptide SapB, and is therefore important for morphogenesis of the bacterium Streptomyces coelicolor. Like most response regulators, RamR consists of an amino-terminal receiver domain and a carboxy-terminal DNA binding domain. Four of five highly conserved active site residues known to be important in other response regulators are present in RamR: D12, D56 (the predicted site of phosphorylation), T84 and K105. Here, we show that in spite of this, RamR did not demonstrate an ability to autophosphorylate in vitro in the presence of small molecule phosphodonors. The unphosphorylated protein behaved as a dimer and bound cooperatively to three sites in the ramC promoter, one with very high affinity and two with lower affinity. On its own, the RamR DNA binding domain could not bind DNA but was able to interfere with the action of full length RamR in a manner suggesting direct protein-protein contact. Surprisingly, substitution of residues D12 or T84 had no effect on RamR function in vivo. In contrast, D56A and K105A substitutions caused defects in both dimer formation and DNA binding while the more conservative substitution, D56N permitted dimer formation but not DNA binding. L102 in RamR corresponds to a well-conserved tyrosine (or aromatic) residue that is important for function in the other response regulators. While a L102Y variant, which introduced the aromatic side-chain usually found at this position, functioned normally, L102A and L102W substitutions blocked RamR function in vivo. We show that these substitutions specifically impaired cooperative DNA binding by RamR at the lower affinity recognition sequences. The biochemical properties of RamR therefore differ markedly from those of other well-characterized response regulators.     

Characterization of a response regulator protein that binds to Anabaena sp. strain L-31 kdp-promoter region

The potassium dependent adenosine triphosphatase (Kdp-ATPase), encoded by the kdp operon, is a potassium uptake system found in several prokaryotes. The cyanobacterium Anabaena sp. strain L-31 shows the presence of two kdp operons (kdp1 and kdp2) of which the kdp2 is predominantly induced in response to potassium limitation or desiccation stress. Two ORFs, encoding a sensor kinase and a response regulator, respectively, were identified upstream of the kdp2 operon in Anabaena sp. strain L-31. The response regulator protein, tagged with 6 additional C-terminal histidine residues, was over-expressed in Escherichia coli and purified by affinity chromatography. Employing the protein-specific antiserum, the response regulator protein was detected in Anabaena L-31 cytosolic fractions. The response regulator protein bound to the kdp2 promoter region with higher affinity than kdp1 promoter region. Addition of acetyl phosphate increased the ability of the protein to bind to kdp2 promoter region by several fold, suggesting a phosphorylation-dependent binding of response regulator to the promoter. These data implicate the response regulator protein in regulation of kdp2 expression in Anabaena sp. strain L-31.     

Coupling of protein localization and cell movements by a dynamically localized response regulator in Myxococcus xanthus

Myxococcus xanthus cells harbor two motility machineries, type IV pili (Tfp) and the A-engine. During reversals, the two machineries switch polarity synchronously. We present a mechanism that synchronizes this polarity switching. We identify the required for motility response regulator (RomR) as essential for A-motility. RomR localizes in a bipolar, asymmetric pattern with a large cluster at the lagging cell pole. The large RomR cluster relocates to the new lagging pole in parallel with cell reversals. Dynamic RomR localization is essential for cell reversals, suggesting that RomR relocalization induces the polarity switching of the A-engine. The analysis of RomR mutants shows that the output domain targets RomR to the poles and the receiver domain is essential for dynamic localization. The small GTPase MglA establishes correct RomR polarity, and the Frz two-component system regulates dynamic RomR localization. FrzS localizes with Tfp at the leading pole and relocates in an Frz-dependent manner to the opposite pole during reversals; FrzS and RomR localize and oscillate independently. The Frz system synchronizes these oscillations and thus the synchronous polarity switching of the motility machineries.     

Fibin, a novel secreted lateral plate mesoderm signal, is essential for pectoral fin bud initiation in zebrafish

We identified a novel secreted protein, fibin, in zebrafish, mice and humans. We inhibited its function in zebrafish embryos by injecting antisense fibin morpholino oligonucleotides. A knockdown of fibin function in zebrafish resulted in no pectoral fin bud initiation and abolished the expression of tbx5, which is involved in the specification of pectoral fin identification. The lack of pectoral fins in fibin-knockdown embryos was partially rescued by injection of fibin RNA. fibin was expressed in the lateral plate mesoderm of the presumptive pectoral fin bud regions. Its expression region was adjacent to that of tbx5. fibin expression temporally preceded tbx5 expression in presumptive pectoral fin bud regions, and not abolished in tbx5-knockdown presumptive fin bud regions. In contrast, fibin expression was abolished in retinoic acid signaling-inhibited or wnt2b-knockdown presumptive fin bud regions. These results indicate that fibin is a secreted signal essential for pectoral fin bud initiation in that it potentially acts downstream of retinoic acid and wnt signaling and is essential for tbx5 expression. The present findings have revealed a novel secreted lateral plate mesoderm signal essential for fin initiation in the lateral plate mesoderm.     

The APC regulator CDH1 is essential for the progression of embryonic cell cycles in Xenopus

The orderly progression of cell cycle depends on timely destruction of key regulators through ubiquitin-mediated proteolysis. The anaphase-promoting complex (APC) is a major component of this degradation machinery and its activation is regulated by CDC20 and CDH1. We demonstrate here that CDH1 mRNA is ubiquitously expressed in Xenopus embryos of all developmental stages. Loss of CDH1 function during early embryonic cell cycles leads to an immediate and prolonged arrest with low cyclin-dependent kinase activity. In contrast, ectopic overexpression of CDH1 induces cell cycle arrest during the first G(1) phase at the midblastula transition. CDH1-dependent degradation of cyclin A is likely involved in this G(1) arrest. Our findings establish the essential roles of CDH1 in embryonic cell cycles.     

The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells

In Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell-cell interactions within the developing Drosophila ventral nerve cord.     

The response regulator Npun_F1278 is essential for scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133

Following exposure to long‐wavelength ultraviolet radiation (UVA), some cyanobacteria produce the indole‐alkaloid sunscreen scytonemin. The genomic region associated with scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme includes 18 cotranscribed genes. A two‐component regulatory system (Npun_F1277/Npun_F1278) directly upstream from the biosynthetic genes was identified through comparative genomics and is likely involved in scytonemin regulation. In this study, the response regulator (RR), Npun_F1278, was evaluated for its ability to regulate scytonemin biosynthesis using a mutant strain of N. punctiforme deficient in this gene, hereafter strain Δ1278. Following UVA radiation, the typical stimulus to initiate scytonemin biosynthesis, Δ1278 was incapable of producing scytonemin. A phenotypic characterization of Δ1278 suggests that aside from the ability to produce scytonemin, the deletion of the Npun_F1278 gene does not affect the cellular morphology, cellular differentiation capability, or lipid‐soluble pigment complement of Δ1278 compared to the wildtype. The mutant, however, had a slower specific growth rate under white light and produced ~2.5‐fold more phycocyanin per cell under UVA than the wildtype. Since Δ1278 does not produce scytonemin, this study demonstrates that the RR gene, Npun_F1278, is essential for scytonemin biosynthesis in N. punctiforme. While most of the evaluated effects of this gene appear to be specific for scytonemin, this regulator may also influence the overall health of the cell and phycobiliprotein synthesis, directly or indirectly. This is the first study to identify a regulatory gene involved in the biosynthesis of the sunscreen scytonemin and posits a link between cell growth, pigment synthesis, and sunscreen production.     

DegU调控单核细胞增生李斯特菌感染宿主细胞及适应高温的机制研究

【目的】本研究旨在探究孤儿调节因子DegU在介导单核细胞增生李斯特菌(Listeria monocytogenes)宿主感染和高温环境适应性方面的调控机制。【方法】本研究以单增李斯特菌参考菌株EGD-e、degU基因缺失菌株ΔdegU和回补菌株CΔdegU为研究材料,通过细胞模型、实时荧光定量聚合酶链式反应和凝胶阻滞试验等方法探究DegU对单增李斯特菌感染宿主细胞和适应高温的调控机制。【结果】研究结果表明：缺失degU后,单增李斯特菌在Caco-2上的黏附和侵袭能力显著降低,在RAW264.7中的增殖能力显著降低,在L929中的空斑形成能力也显著降低;进一步通过实时荧光定量聚合酶链式反应检测degU基因缺失后引起的单增李斯特菌毒力因子转录水平变化,发现多个重要毒力因子转录水平均显著下调;该试验结果还发现与毒力相关的热应激基因clpE(受CtsR抑制的ATP依赖蛋白水解酶编码基因)转录水平显著升高,而在43℃高温条件下,clpE转录水平显著降低;进一步通过凝胶阻滞试验结果表明DegU能够与clpE的启动子直接结合。【结论】综上所述,degU基因缺失能够降低单增李斯特菌在宿主感染过程中的细...     

The crystal structure of YloQ, a circularly permuted GTPase essential for Bacillus subtilis viability

yloQ is one of 11 essential genes in Bacillus subtilis with unknown roles in the physiology of the cell. It encodes a polypeptide of 298 residues with motifs characteristic of GTPases. As a contribution to elucidating its indispensable cellular function, we have solved the crystal structure of YloQ to 1.6 A spacing, revealing a three-domain organisation. At the heart of the molecule is the putative GTPase domain, which exhibits a classical alpha/beta nucleotide-binding fold with a topology very similar to that of Ras and Era. However, as anticipated from the order in which the conserved G protein motifs appear in the sequence, the GTPase domain fold in YloQ is circularly permuted with respect to the classical GTPases. The nucleotide-binding pocket in YloQ is unoccupied, and analysis of the phosphate-binding (P) loop indicates that conformational changes in this region would be needed to accommodate GTP. The GTPase domain is flanked at its N terminus by a beta-barrel domain with an oligonucleotide/oligosaccharide-binding (OB) fold, and at its C terminus by an alpha-helical domain containing a coordinated zinc ion. This combination of protein modules is unique to YloQ and its orthologues. Sequence comparisons reveal a clustering of conserved basic and aromatic residues on one face of the OB domain, perhaps pointing to a role for YloQ in nucleic acid binding. The zinc ion in the alpha-helical domain is coordinated by three cysteine residues and a histidine residue in a novel ligand organisation. The juxtaposition of the switch I and switch II regions of the G domain and the OB and zinc-binding domains suggests that chemical events at the GTPase active site may be transduced into relative movements of these domains. The pattern of conserved residues and electrostatic surface potential calculations suggest that the OB and/or Zn-binding domains participate in nucleic acid binding consistent with a possible role for YloQ at some stage during mRNA translation.     

The yeast Coq4 polypeptide organizes a mitochondrial protein complex essential for coenzyme Q biosynthesis

Coenzyme Q is a redox active lipid essential for aerobic respiration. The Coq4 polypeptide is required for Q biosynthesis and growth on non-fermentable carbon sources, however its exact function in this pathway is not known. Here we probe the functional roles of Coq4p in a yeast Q biosynthetic polypeptide complex. A yeast coq4-1 mutant harboring an E226K substitution is unable to grow on nonfermentable carbon sources. The coq4-1 yeast mutant retains significant Coq3p O-methyltransferase activity, and mitochondria isolated from coq4-1 and coq4-2 (E₁₂₁K) yeast point mutants contain normal steady state levels of Coq polypeptides, unlike the decreased levels of Coq polypeptides generally found in strains harboring coq gene deletions. Digitonin-solubilized mitochondrial extracts prepared from yeast coq4 point mutants show that Coq3p and Coq4 polypeptides no longer co-migrate as high molecular mass complexes by one- and two-dimensional Blue Native-PAGE. Similarly, gel filtration chromatography confirms that O-methyltransferase activity, Coq3p, Coq4p, and Coq7p migration are disorganized in the coq4-1 mutant mitochondria. The data suggest that Coq4p plays an essential role in organizing a Coq enzyme complex required for Q biosynthesis.     

The signaling pathway in histidine kinase and the response regulator complex revealed by X-ray crystallography and solution scattering

The structure of a histidine kinase (ThkA) complexed with a response regulator (TrrA) in the two-component regulatory system from hyperthermophile Thermotoga maritima was determined by a combination of X-ray crystallography at a resolution of 4.2 A and small-angle X-ray scattering (SAXS). The boundary of the three component domains (PAS-sensor, dimerization and catalytic domains) of ThkA and the bound TrrA molecule were unambiguously assigned in the electron density map at 4.2 A resolution. ThkA forms a dimer with crystallographic 2-fold symmetry and two monomeric TrrAs bind to the ThkA dimer. SAXS experiments also confirmed this association state in solution and specific binding between ThkA and TrrA (Kd=8.2x10(-11) M(-2)). The association interface between ThkA and TrrA contains the phosphotransfer His residue in the ThkA, indicative of an efficient receipt of the phosphoryl group. One Per-Arnt-Sim (PAS) domain does not interact with the other PAS domain, but with the catalytic domain of the same polypeptide chain and with one TrrA molecule. Observed inter-domain and inter-molecular interactions reveal a definite pathway of signal transduction in the kinase/regulator complex. In addition, we propose a responsible role of TrrA for the feedback regulation of sensing and/or kinase activities of ThkA.     

The Dlx5 homeobox gene is essential for vestibular morphogenesis in the mouse embryo through a BMP4-mediated pathway

In the mouse embryo, Dlx5 is expressed in the otic placode and vesicle, and later in the semicircular canals of the inner ear. In mice homozygous for a null Dlx5/LacZ allele, a severe dysmorphogenesis of the vestibular region is observed, characterized by the absence of semicircular canals and the shortening of the endolymphatic duct. Minor defects are observed in the cochlea, although Dlx5 is not expressed in this region. Cristae formation is severely impaired; however, sensory epithelial cells, recognized by calretinin immunostaining, are present in the vestibular epithelium of Dlx5(-/-) mice. The maculae of utricle and saccule are present but cells appear sparse and misplaced. The abnormal morphogenesis of the semicircular canals is accompanied by an altered distribution of proliferating and apoptotic cells. In the Dlx5(-/-) embryos, no changes in expression of Nkx5.1(Hmx3), Pax2, and Lfng have been seen, while expression of bone morphogenetic protein-4 (Bmp4) was drastically reduced. Notably, BMP4 has been shown to play a fundamental role in vestibular morphogenesis of the chick embryo. We propose that development of the semicircular canals and the vestibular inner ear requires the independent control of several homeobox genes, which appear to exert their function via tight regulation of BPM4 expression and the regional organization of cell differentiation, proliferation, and apoptosis.     

The N-terminal prodomain of sV23 is essential for the assembly of a functional vitelline membrane network in Drosophila

The vitelline membrane is a specialized extracellular matrix that surrounds and protects the oocyte. Recent studies indicate that it also serves as a storage site for embryonic pattern determinants. sV23, a major vitelline membrane protein, is essential for the morphogenesis of the vitelline membrane as sV23 protein null mutants lay flaccid, infertile eggs. By analyzing a series of sV23 mutant transgenes in the sV23 protein null genetic background, we have shown that sV23 is secreted as a proprotein in functional excess and that C- and N-terminal prodomains are removed successively, following its deposition in the extracellular space. Although a target site for subtilisin-like convertases is essential for N-terminal processing, N-terminal processing is not necessary for the assembly of a functional vitelline membrane layer. While C-terminal truncations were tolerated, the removal of N-terminal sequences lead to the production of flaccid, infertile eggs with a soluble, rather than insoluble, vitelline membrane network. We propose that the hydrophobic N-terminal prodomain plays an early and essential role in aligning molecules within the vitelline membrane network, much like hydrophobic domains within elastin drive the assembly and alignment of molecules within elastin-based extracellular matrices.     

The Grb2/PLD2 interaction is essential for lipase activity, intracellular localization and signaling in response to EGF

The adaptor protein Grb2 associates with phospholipase D2 (PLD2), but it is not known if this interaction is necessary for the functionality of the lipase in vivo. We demonstrate that stable short hairpin RNA (shRNA)-based silencing of Grb2, a critical signal transducer of the epidermal growth factor receptor (EGFR) and linker to the Ras/Erk pathway, resulted in the reduction of PLD2 activity in COS7 cells. Transfection of a Grb2 construct refractory to shGrb2 silencing (XGrb2(SiL)) into the Grb2-knockdown cells (COS7(shGrb2)), resulted in the nearly full rescue of PLD2 activity. However, Grb2-R86K, an SH2-deficient mutant of Grb2 that is incapable of binding to PLD2, failed to induce an enhancement of the impaired PLD2 activity in COS7(shGrb2) cells. Grb2 and PLD2 are directly associated and Grb2 is brought down with anti-myc antibodies irrespective of the presence or absence of EGFR activation. Immunofluorescence microscopy showed that co-transfected PLD2 and Grb2 re-localize to Golgi-like structures after EGF stimulation. Since this was not observed in cotransfection experiments with Grb2 and PLD2-Y169/179F, a lipase mutant that does not bind to Grb2, we inferred that Grb2 serves to hijack PLD2 to the perinuclear Golgi region through its SH2 domain. Supporting this is the finding that the primary cell line HUVEC expresses PLD2 diffusely in the cytoplasm and in the perinuclear Golgi region, where PLD2 and Grb2 colocalize. Such colocalization in primary cells increased after stimulation with EGF. These results demonstrate for the first time that the presence of Grb2 and its interaction with localized intracellular structures is essential for PLD2 activity and signaling in vivo.     

An internal hydrophobic helical domain of Bacillus subtilis enolase is essential but not sufficient as a non-cleavable signal for its secretion

Many cytoplasmic proteins without a cleavable signal peptide, including enolase, are secreted during the stationary phase in Bacillus subtilis but the molecular mechanism is not yet clear. We previously identified a highly conserved embedded membrane domain in an internal hydrophobic α-helix of enolase that plays an important role in its secretion. In this study, we examined the role of the helix in more detail for the secretion of enolase. Altering this helix by mutations showed that many mutated forms in this domain were not secreted, some of which were not stable as a soluble form in the cytoplasm. On the other hand, mutations on the flanking regions of the helix or the conserved basic residues showed no deleterious effect. Bacillus enolase with the proper hydrophobic helical domain was also exported extracellularly in Escherichia coli, indicating that the requirement of the helix for the secretion of enolase is conserved in these species. GFP fusions with enolase regions showed that the hydrophobic helix domain itself was not sufficient to serve as a functional secretion signal; a minimal length of N-terminus 140 amino acids was required to mediate the secretion of the fused reporter GFP. We conclude that the internal hydrophobic helix of enolase is essential but is not sufficient as a signal for secretion; the intact long N-terminus including the hydrophobic helix domain is required to serve as a non-cleavable signal for the secretion of Bacillus enolase.