Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress.

As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.

Molecular keys to speciation: DNA polymorphism and the control of genetic exchange in enterobacteria

Speciation involves the establishment of genetic barriers between closely related organisms. The extent of genetic recombination is a key determinant and a measure of genetic isolation. The results reported here reveal that genetic barriers can be established, eliminated, or modified by manipulating two systems which control genetic recombination, SOS and mismatch repair. The extent of genetic isolation between enterobacteria is a simple mathematical function of DNA sequence divergence. The function does not depend on hybrid DNA stability, but rather on the number of blocks of sequences identical in the two mating partners and sufficiently large to allow the initiation of recombination. Further, there is no obvious discontinuity in the function that could be used to define a level of divergence for distinguishing species.

Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti

Succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti, is composed of polymerized octasaccharide subunits, each of which consists of one galactose and seven glucoses with succinyl, acetyl, and pyruvyl modifications. Production of specific low molecular weight forms of R. meliloti exported and surface polysaccharides, including succinoglycan, appears to be important for nodule invasion. In a previous study of the roles of the various exo gene products in succinoglycan biosynthesis, exoP, exoQ, and exoT mutants were found to synthesize undecaprenol-linked fully modified succinoglycan octasaccharide subunits, suggesting possible roles for their gene products in polymerization or transport. Using improved techniques for analyzing succinoglycan biosynthesis by these mutants, we have obtained evidence indicating that R. meliloti has genetically separable systems for the synthesis of high molecular weight succinoglycan and the synthesis of a specific class of low molecular weight oligosaccharides consisting of dimers and trimers of the octasaccharide subunit. Models to account for our unexpected findings are discussed. Possible roles for the ExoP, ExoQ, and ExoT proteins are compared and contrasted with roles that have been suggested on the basis of homologies to key proteins involved in the biosynthesis of O-antigens and of certain exported or capsular cell surface polysaccharides.

Molecular Interaction Map of the Mammalian Cell Cycle Control and DNA Repair Systems

Eventually to understand the integrated function of the cell cycle regulatory network, we must organize the known interactions in the form of a diagram, map, and/or database. A diagram convention was designed capable of unambiguous representation of networks containing multiprotein complexes, protein modifications, and enzymes that are substrates of other enzymes. To facilitate linkage to a database, each molecular species is symbolically represented only once in each diagram. Molecular species can be located on the map by means of indexed grid coordinates. Each interaction is referenced to an annotation list where pertinent information and references can be found. Parts of the network are grouped into functional subsystems. The map shows how multiprotein complexes could assemble and function at gene promoter sites and at sites of DNA damage. It also portrays the richness of connections between the p53-Mdm2 subsystem and other parts of the network.

Src, a molecular switch governing gain control of synaptic transmission mediated by N-methyl-d-aspartate receptors

The N-methyl-d-aspartate (NMDA) receptor is a principal subtype of glutamate receptor mediating fast excitatory transmission at synapses in the dorsal horn of the spinal cord and other regions of the central nervous system. NMDA receptors are crucial for the lasting enhancement of synaptic transmission that occurs both physiologically and in pathological conditions such as chronic pain. Over the past several years, evidence has accumulated indicating that the activity of NMDA receptors is regulated by the protein tyrosine kinase, Src. Recently it has been discovered that, by means of up-regulating NMDA receptor function, activation of Src mediates the induction of the lasting enhancement of excitatory transmission known as long-term potentiation in the CA1 region of the hippocampus. Also, Src has been found to amplify the up-regulation of NMDA receptor function that is produced by raising the intracellular concentration of sodium. Sodium concentration increases in neuronal dendrites during high levels of firing activity, which is precisely when Src becomes activated. Therefore, we propose that the boost in NMDA receptor function produced by the coincidence of activating Src and raising intracellular sodium may be important in physiological and pathophysiological enhancement of excitatory transmission in the dorsal horn of the spinal cord and elsewhere in the central nervous system.

Ultrafast detection and control of molecular dynamics

Many elementary chemical and physical processes such as the breaking of a chemical bond or the vibrational motion of atoms within a molecule take place on a femtosecond (fs = 10−15 s) or picosecond (ps = 10−12 s) time scale. It is now possible to monitor these events as a function of time with temporal resolution well below 100 fs. This capability is based on the pump-probe technique where one optical pulse triggers a reaction and a second delayed optical pulse probes the changes that ensue. To illustrate this capability, the dynamics of ligand motion within a protein are presented. Moving beyond casual observation of a reaction to active control of its outcome requires additional experimental and theoretical effort. To illustrate the concept of control, the effect of optical pulse duration on the vibrational dynamics of a tri-atomic molecule are discussed. The experimental and theoretical resources currently available are poised to make the dream of reaction control a reality for certain molecular systems.

Engineering precision RNA molecular switches

Ligand-specific molecular switches composed of RNA were created by coupling preexisting catalytic and receptor domains via structural bridges. Binding of ligand to the receptor triggers a conformational change within the bridge, and this structural reorganization dictates the activity of the adjoining ribozyme. The modular nature of these tripartite constructs makes possible the rapid construction of precision RNA molecular switches that trigger only in the presence of their corresponding ligand. By using similar enzyme engineering strategies, new RNA switches can be made to operate as designer molecular sensors or as a new class of genetic control elements.

A single point mutation in the pore region of the epithelial Na+ channel changes ion selectivity by modifying molecular sieving

The epithelial Na+ channel (ENaC) belongs to a new class of channel proteins called the ENaC/DEG superfamily involved in epithelial Na+ transport, mechanotransduction, and neurotransmission. The role of ENaC in Na+ homeostasis and in the control of blood pressure has been demonstrated recently by the identification of mutations in ENaC β and γ subunits causing hypertension. The function of ENaC in Na+ reabsorption depends critically on its ability to discriminate between Na+ and other ions like K+ or Ca2+. ENaC is virtually impermeant to K+ ions, and the molecular basis for its high ionic selectivity is largely unknown. We have identified a conserved Ser residue in the second transmembrane domain of the ENaC α subunit (αS589), which when mutated allows larger ions such as K+, Rb+, Cs+, and divalent cations to pass through the channel. The relative ion permeability of each of the αS589 mutants is related inversely to the ionic radius of the permeant ion, indicating that αS589 mutations increase the molecular cutoff of the channel by modifying the pore geometry at the selectivity filter. Proper geometry of the pore is required to tightly accommodate Na+ and Li+ ions and to exclude larger cations. We provide evidence that ENaC discriminates between cations mainly on the basis of their size and the energy of dehydration.

Modeling the control of DNA replication in fission yeast

A central event in the eukaryotic cell cycle is the decision to commence DNA replication (S phase). Strict controls normally operate to prevent repeated rounds of DNA replication without intervening mitoses (“endoreplication”) or initiation of mitosis before DNA is fully replicated (“mitotic catastrophe”). Some of the genetic interactions involved in these controls have recently been identified in yeast. From this evidence we propose a molecular mechanism of “Start” control in Schizosaccharomyces pombe. Using established principles of biochemical kinetics, we compare the properties of this model in detail with the observed behavior of various mutant strains of fission yeast: wee1− (size control at Start), cdc13Δ and rum1OP (endoreplication), and wee1− rum1Δ (rapid division cycles of diminishing cell size). We discuss essential features of the mechanism that are responsible for characteristic properties of Start control in fission yeast, to expose our proposal to crucial experimental tests.

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5′ upstream region of the grisea ORF. One mutation is a G → A transition at the 5′ splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

pH gating of ROMK (Kir1.1) channels: Control by an Arg-Lys-Arg triad disrupted in antenatal Bartter syndrome

Inward-rectifier K+ channels of the ROMK (Kir1.1) subtype are responsible for K+ secretion and control of NaCl absorption in the kidney. A hallmark of these channels is their gating by intracellular pH in the neutral range. Here we show that a lysine residue close to TM1, identified previously as a structural element required for pH-induced gating, is protonated at neutral pH and that this protonation drives pH gating in ROMK and other Kir channels. Such anomalous titration of this lysine residue (Lys-80 in Kir1.1) is accomplished by the tertiary structure of the Kir protein: two arginines in the distant N and C termini of the same subunit (Arg-41 and Arg-311 in Kir1.1) are located in close spatial proximity to the lysine allowing for electrostatic interactions that shift its pKa into the neutral pH range. Structural disturbance of this triad as a result from a number of point mutations found in patients with antenatal Bartter syndrome shifts the pKa of the lysine residue off the neutral pH range and results in channels permanently inactivated under physiological conditions. Thus, the results provide molecular understanding for normal pH gating of Kir channels as well as for the channel defects found in patients with antenatal Bartter syndrome.

Molecular characterization of a mutable pigmentation phenotype and isolation of the first active transposable element from Sorghum bicolor

Accumulation of red phlobaphene pigments in sorghum grain pericarp is under the control of the Y gene. A mutable allele of Y, designated as y-cs (y-candystripe), produces a variegated pericarp phenotype. Using probes from the maize p1 gene that cross-hybridize with the sorghum Y gene, we isolated the y-cs allele containing a large insertion element. Our results show that the Y gene is a member of the MYB-transcription factor family. The insertion element, named Candystripe1 (Cs1), is present in the second intron of the Y gene and shares features of the CACTA superfamily of transposons. Cs1 is 23,018 bp in size and is bordered by 20-bp terminal inverted repeat sequences. It generated a 3-bp target site duplication upon insertion within the Y gene and excised from y-cs, leaving a 2-bp footprint in two cases analyzed. Reinsertion of the excised copy of Cs1 was identified by Southern hybridization in the genome of each of seven red pericarp revertant lines tested. Cs1 is the first active transposable element isolated from sorghum. Our analysis suggests that Cs1-homologous sequences are present in low copy number in sorghum and other grasses, including sudangrass, maize, rice, teosinte, and sugarcane. The low copy number and high transposition frequency of Cs1 imply that this transposon could prove to be an efficient gene isolation tool in sorghum.

Effects of Age on the Posttranscriptional Regulation of CCAAT/Enhancer Binding Protein α and CCAAT/Enhancer Binding Protein β Isoform Synthesis in Control and LPS-Treated Livers

The CCAAT/enhancer binding protein α (C/EBPα) and CCAAT/enhancer binding protein β (C/EBPβ) mRNAs are templates for the differential translation of several isoforms. Immunoblotting detects C/EBPαs with molecular masses of 42, 38, 30, and 20 kDa and C/EBPβs of 35, 20, and ∼8.5 kDa. The DNA-binding activities and pool levels of p42C/EBPα and p30C/EBPα in control nuclear extracts decrease significantly whereas the binding activity and protein levels of the 20-kDa isoforms increase dramatically with LPS treatment. Our studies suggest that the LPS response involves alternative translational initiation at specific in-frame AUGs, producing specific C/EBPα and C/EBPβ isoform patterns. We propose that alternative translational initiation occurs by a leaky ribosomal scanning mechanism. We find that nuclear extracts from normal aged mouse livers have decreased p42C/EBPα levels and binding activity, whereas those of p20C/EBPα and p20C/EBPβ are increased. However, translation of 42-kDa C/EBPα is not down-regulated on polysomes, suggesting that aging may affect its nuclear translocation. Furthermore, recovery of the C/EBPα- and C/EBPβ-binding activities and pool levels from an LPS challenge is delayed significantly in aged mouse livers. Thus, aged livers have altered steady-state levels of C/EBPα and C/EBPβ isoforms. This result suggests that normal aging liver exhibits characteristics of chronic stress and a severe inability to recover from an inflammatory challenge.

Control of Cyclin D1, p27Kip1, and Cell Cycle Progression in Human Capillary Endothelial Cells by Cell Shape and Cytoskeletal Tension

The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that were prevented from spreading in medium containing soluble growth factors exhibited normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells failed to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlated with a failure to increase cyclin D1 protein levels, down-regulate the cell cycle inhibitor p27Kip1, and phosphorylate the retinoblastoma protein in late G1. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.

A global two component signal transduction system that integrates the control of photosynthesis, carbon dioxide assimilation, and nitrogen fixation

Photosynthesis, biological nitrogen fixation, and carbon dioxide assimilation are three fundamental biological processes catalyzed by photosynthetic bacteria. In the present study, it is shown that mutant strains of the nonsulfur purple photosynthetic bacteria Rhodospirillum rubrum and Rhodobacter sphaeroides, containing a blockage in the primary CO2 assimilatory pathway, derepress the synthesis of components of the nitrogen fixation enzyme complex and abrogate normal control mechanisms. The absence of the Calvin–Benson–Bassham (CBB) reductive pentose phosphate CO2 fixation pathway removes an important route for the dissipation of excess reducing power. Thus, the mutant strains develop alternative means to remove these reducing equivalents, resulting in the synthesis of large amounts of nitrogenase even in the presence of ammonia. This response is under the control of a global two-component signal transduction system previously found to regulate photosystem biosynthesis and the transcription of genes required for CO2 fixation through the CBB pathway and alternative routes. In addition, this two-component system directly controls the ability of these bacteria to grow under nitrogen-fixing conditions. These results indicate that there is a molecular link between the CBB and nitrogen fixation process, allowing the cell to overcome powerful control mechanisms to remove excess reducing power generated by photosynthesis and carbon metabolism. Furthermore, these results suggest that the two-component system integrates the expression of genes required for the three processes of photosynthesis, nitrogen fixation, and carbon dioxide fixation.