Iron-containing Proteins Research Articles

Small RNA ArrF Regulates the Expression of sodB and feSII Genes in Azotobacter vinelandii

Azotobacter vinelandii contains a prrF-like sequence in a noncoding region of the chromosome. Like the Pseudomonas aeruginosa PrrF small RNA-encoding genes, the expression of the sequence, herein named arrF (Azotobacter regulatory RNA involving Fe), was increased 100-fold in wild-type cells in response to iron depletion. The level of ArrF was also increased to the same degree in the iron-replete fur mutant, but down back to a wild-type level when this fur mutant was complemented with the wild-type fur gene. These results, with the location of arrF gene in a noncoding region, suggest that this gene encodes an iron-responsive small RNA whose expression is negatively regulated by the Fur-Fe(2+) complex. Disruption of this arrF gene upregulated the expression of iron-containing superoxide dismutase and FeSII protein, whereas fur mutation or iron depletion decreased the level of their transcript. A short region in the 5'-untranslated region of each transcript was predicted to be quite complementary to the core sequence of ArrF, assuming that ArrF represses the expression of the genes under Fur control by an antisense RNA mechanism. However, unlike the P. aeruginosa PrrF that has extensive targets in the tricarboxylic acid cycle and glyoxylate cycle, ArrF had little effect on those genes. The findings that there is a poor overlap between ArrF and PrrF targets and that the FeSII gene, which is present only in the chromosome of nitrogen-fixing bacterial species, is controlled by ArrF suggest that ArrF might have unique targets, some of which are involved in N(2) fixation.

The Bacillus subtilis iron-sparing response is mediated by a Fur-regulated small RNA and three small, basic proteins

Regulation of bacterial iron homeostasis is often controlled by the iron-sensing ferric uptake repressor (Fur). The Bacillus subtilis Fur protein acts as an iron-dependent repressor for siderophore biosynthesis and iron transport proteins. Here, we demonstrate that Fur also coordinates an iron-sparing response that acts to repress the expression of iron-rich proteins when iron is limiting. When Fur is inactive, numerous iron-containing proteins are down-regulated, including succinate dehydrogenase, aconitase, cytochromes, and biosynthetic enzymes for heme, cysteine, and branched chain amino acids. As a result, a fur mutant grows slowly in a variety of nutrient conditions. Depending on the growth medium, rapid growth can be restored by mutations in one or more of the molecular effectors of the iron-sparing response. These effectors include the products of three Fur-regulated operons that encode a small RNA (FsrA) and three small, basic proteins (FbpA, FbpB, and FbpC). Extensive complementarity between FsrA and the leader region of the succinate dehydrogenase operon is consistent with an RNA-mediated translational repression mechanism for this target. Thus, iron deprivation in B. subtilis activates pathways to remodel the proteome to preserve iron for the most critical cellular functions.

Three Consecutive Arginines Are Important for the Mycobacterial Peptide Deformylase Enzyme Activity

Genes encoding the peptide deformylase enzyme (def) are present in all eubacteria and are involved in the deformylation of the N-formyl group of newly synthesized polypeptides during protein synthesis. We compared the amino acid sequences of this enzyme in different mycobacterial species and found that they are highly conserved (76% homology with 62% identity); however, when this comparison was extended to other eubacterial homologs, it emerged that the mycobacterial proteins have an insertion region containing three consecutive arginine residues (residues 77-79 in Mycobacterium tuberculosis peptide deformylase (mPDF)). Here, we demonstrate that these three arginines are important for the activity of mPDF. Circular dichroism studies of wild-type mPDF and of mPDF containing individual conservative substitutions (R77K, R78K, or R79K) or combined substitutions incorporated into a triple mutant (R77K/R78K/R79K) indicate that such mutations cause mPDF to undergo structural alterations. Molecular modeling of mPDF suggests that the three arginines are distal to the active site. Molecular dynamics simulations of wild-type and mutant mPDF structures indicate that the arginines may be involved in the stabilization of substrate binding pocket residues for their proper interaction with peptide(s). Treatment with 5'-phosphothiorate-modified antisense oligodeoxyribonucleotides directed against different regions of def from M. tuberculosis inhibits growth of Mycobacterium smegmatis in culture. Taken together, these results hold out the possibility of future design of novel mycobacteria-specific PDF inhibitors.

Hemoglobin in the Kidney

Hemoglobins (Hb) in both prokaryotes and eukaryotes belong to an ancient family of heme-associated, iron-containing globin proteins. Mammalian Hb are tetrameric metalloproteins (approximately 68 kD) that are present in millimolar concentrations within red blood cells, where they function as O2-

Proteomic analysis of Helicobacter pylori cellular proteins fractionated by ammonium sulfate precipitation

Among 1590 ORFs in the Helicobacter pylori genome, >250 have been identified as authentic genes by proteomic analysis. Low-abundance proteins need to be enriched to a minimal amount for MALDI-TOF analysis and salt precipitation has generally been used for protein enrichment. Here, a whole-cell extract of H. pylori strain 26695 was subjected to protein fractionation with stepwise concentrations of ammonium sulfate and the proteins were displayed by 2-DE. The protein spots were quantified using PDQUEST software and identified by peptide fingerprinting. The 2-DE profiles and intensities of individual protein spots differed among the protein fractions. Out of the 98 identified proteins, 61 were found in the stepwise ammonium sulfate fractions but not in the whole-cell extract. Out of these, 37 proteins, including KdsA, were found exclusively in a single fraction. In contrast, GroEL, UreA, UreB, TrxA, NapA, and FldA were ubiquitously present in all fractions. Iron-containing proteins such as NapA, SodB, CeuE, and Pfr were found predominantly in the 100% saturated ammonium sulfate precipitate. Additionally, 29 proteins were newly identified in this study. These data will facilitate the preparation of significant H. pylori proteins, as well as provide information about low-abundance proteins.

Bioinorganic chemistry: a short course

Chapter 1: Inorganic Chemistry Essentials. Introduction . Essential Chemical Elements. Metals in Biological Systems: A Survey. Inorganic Chemistry Basics. Biological Metal Ion Complexation. Thermodynamics. Kinetics. Electronic and Geometric Structures of Metals in Biological Systems. Bioorganometallic Chemistry. Electron Transfer. Introduction to Biological Metal Homeostasis, Transport, and Storage. Chapter 2: Biochemistry Fundamentals. Introduction. Proteins. Amino Acid Building Blocks. Protein Structure. Protein Sequencing and Proteomics. Protein Function, Enzymes and Enzyme Kinetics. Nucleic Acids. DNA and RNA Building Blocks. DNA and RNA Molecular Structures. Transmission of Genetic Information. Genetic Mutations and Site-Directed Mutagenesis. Genes and Cloning. Genomics and the Human Genome. The ATP-binding cassette (ABC) Transporter Superfamily. Chapter 3: Instrumental Methods. Introduction. Analytical Instrument-Based Methods. Spectroscopy. X-ray Absorption Spectroscopy (XAS) and Extended X-ray Absorption Fine Structure (EXAFS). Theoretical Aspects and Hardware. Descriptive Examples. X-ray Crystallography. Introduction. Crystallization and Crystal Habits. Theory and Hardware. Descriptive Examples. Electron Paramagnetic Resonance. Theory and Determination of g-values. Hyperfine and Superhyperfine Interactions. Descriptive Examples. Nuclear Magnetic Resonance. Theoretical Aspects. Nuclear Screening and the Chemical Shift. Spin-Spin coupling. Techniques of Spectral Integration and Spin-Spin Decoupling. Nuclear Magnetic Relaxation. The nuclear Overhauser Effect (NOE). Obtaining the NMR spectrum. Two-dimensional NMR Spectroscopy. Correlation Spectroscopy (COSY). Nuclear Overhauser Effect Spectroscopy (NOESY). Descriptive Examples. Mossbauer Spectroscopy. Theoretical Aspects. Quadrupole Splitting and the Isomer Shift. Magnetic Hyperfine Interactions. Descriptive Examples. Atomic Force Microscopy. Fast Methods. Stopped-Flow Kinetic Methods. Flash Photolysis. Time-Resolved Crystallography. Chapter 4: Introduction to Computer-Based Methods. Computer Hardware . Molecular Modeling and Molecular Mechanics. Introduction to MM. Molecular Modeling, Molecular Mechanics and Molecular Dynamics. Biomolecule Modeling. Molecular Modeling Descriptive Examples. Quantum Mechanics-Based Computational Methods. Introduction. Ab-initio Methods. Density Functional Theory. Semi-empirical Methods. Computer Software for Chemistry. Mathematical Software. World Wide Web Online Resources. Nomenclature and Visualization Resources. Online Societies, Literature, Materials, Equipment Webservers. Chapter 5: Group I and II metals in biological systems. Transport and storage of metal ions. Homeostasis involving Na+, K+, Ca2+, Mg2+, Zn2+ (as well as P, Cl, and H). Movement of molecules and ions across membranes. Proton pumps. Ion pumps. Active versus passive transport . Na+/ K+ ATPases. Magnesium dependent biomolecules. Kinases. ATP-pumps. Catalytic RNA and Magnesium. Magnesium in chlorophyll. Calcium dependent biomolecules. Calmodulins and triggering. Biochemistry of nitrogen oxides, nitroxyl, and peroxynitrite ion. Chapter 6: Iron Containing Proteins and Enzymes. Iron-containing proteins with porphyrin ligand systems. Hemoglobin and Myoglobin (first edition material somewhat condensed). Cytochrome P-450: a monooxygenase. Iron-containing cytochromes in photosynthesis. Green plant cytochromes. Cytochrome bc559. Cytochrome bf. Bacterial cytochromes. Cytochrome c2. Cytochrome bc1. Iron-containing cytochromes in the respiratory pathway. Cytochrome c. Cytochrome c oxidase. Non-Heme Iron-containing proteins. MMO-Methane Monooxygenase. Catechol dioxygenases. Iron-oxo clusters. Transferrin for metal transport . Ferritin for metal storage. Hemerythrin. Purple acid phosphatase. Iron-sulfur cluster proteins. Rubredoxin. Ferrodoxins. Aconitase. Chapter 7: Enzymes containing zinc, nickel, and enzymes with multiple metal cofactors. Zinc homeostasis, transport, and storage. Zinc Enzymes. Alkaline phosphatase. Alcohol dehydrogenase. Aspartate transcarbamoylase. Carbonic anhydrase. Carboxypeptidase A and B. Zinc Fingers. Nickel Enzymes. Urease. F430. Enzymes with multiple metal cofactors. Hydrogenases. Carbon monoxide dehydrogenases. Chapter 8 Disease, Disease Diagnosis and Treatment Utilizing Inorganic Species. Metal Transport and Metallochaperones. Disease States. Metal deficiency. Metal toxicity. Copper toxicity. FALS. Wilson and Menkes diseases. Disease Diagnosis. Technetium Imaging Agents. Gadolinium MRI Imaging Agents. Quantum Dots for In vivo Imaging. Metals in Medicine. Manganese superoxide mimetics. Vanadium-based Diabetes Drugs. Platinum-containing Anticancer Agents. Nanomedicine.

Can Lipofuscin Accumulation Be Prevented?

During normal autophagic degradation of mitochondria and iron-containing proteins in lysosomes, iron is released intralysosomally where it may react with hydrogen peroxide forming hydroxyl radicals (Fenton reaction). Depending on their rate of formation, these highly reactive radicals can cross-link intralysosomal material, leading to lipofuscin formation, or destabilize the lysosomal membrane, which induces apoptosis/necrosis. Since the sensitivity of lysosomes to oxidative stress can be manipulated by altering the intralysosomal level of redox-active iron, it follows that lipofuscin formation might also be influenced. It is suggested that pulse doses of iron chelators that easily penetrate membranes could be used to diminish lipofuscinogenesis.

Protocol to determine accurate absorption coefficients for iron-containing transferrins

An accurate protein concentration is an essential component of most biochemical experiments. The simplest method to determine a protein concentration is by measuring the A 280 using an absorption coefficient (ε) and applying the Beer–Lambert law. For some metalloproteins (including all transferrin family members), difficulties arise because metal binding contributes to the A 280 in a nonlinear manner. The Edelhoch method is based on the assumption that the ε of a denatured protein in 6 M guanidine–HCl can be calculated from the number of the tryptophan, tyrosine, and cystine residues. We extend this method to derive ε values for both apo- and iron-bound transferrins. The absorbance of an identical amount of iron-containing protein is measured in (i) 6 M guanidine–HCl (denatured, no iron), (ii) pH 7.4 buffer (nondenatured with iron), and (iii) pH 5.6 (or lower) buffer with a chelator (nondenatured without iron). Because the iron-free apoprotein has an identical A 280 under nondenaturing conditions, the difference between the reading at pH 7.4 and the lower pH directly reports the contribution of the iron. The method is fast and consumes approximately 1 mg of sample. The ability to determine accurate ε values for transferrin mutants that bind iron with a wide range of affinities has proven to be very useful; furthermore, a similar approach could easily be followed to determine ε values for other metalloproteins in which metal binding contributes to the A 280.

‘Iron‐saturated’ lactoferrin is a potent natural adjuvant for augmenting cancer chemotherapy

Bovine lactoferrin (bLf), an iron-containing natural defence protein found in bodily secretions, has been reported to inhibit carcinogenesis and the growth of tumours. Here, we investigated whether natural bLf and iron-saturated forms of bLf differ in their ability to augment cancer chemotherapy. bLf was supplemented into the diet of C57BL/6 mice that were subsequently challenged subcutaneously with tumour cells, and treated by chemotherapy. Chemotherapy eradicated large (0.6 cm diameter) EL-4 lymphomas in mice that had been fed iron-saturated bLf (here designated Lf(+)) for 6 weeks prior to chemotherapy, but surprisingly not in mice that were fed lesser iron-saturated forms of bLf, including apo-bLf (4% iron saturated), natural bLf (approximately 15% iron saturated) and 50% iron-saturated bLf. Lf(+)-fed mice bearing either EL-4, Lewis lung carcinoma or B16 melanoma tumours completely rejected their tumours within 3 weeks following a single injection of either paclitaxel, doxorubicin, epirubicin or fluorouracil, whereas mice fed the control diet were resistant to chemotherapy. Lf(+) had to be fed to mice for more than 2 weeks prior to chemotherapy to be wholly effective in eradicating tumours from all mice, suggesting that it acts as a competence factor. It significantly reduced tumour vascularity and blood flow, and increased antitumour cytotoxicity, tumour apoptosis and the infiltration of tumours by leukocytes. Lf(+) bound to the intestinal epithelium and was preferentially taken up within Peyer's patches. It increased the production of Th1 and Th2 cytokines within the intestine and tumour, including TNF, IFN-gamma, as well as nitric oxide that have been reported to sensitize tumours to chemotherapy. Importantly, it restored both red and white peripheral blood cell numbers depleted by chemotherapy, potentially fortifying the mice against cancer. In summary, bLf is a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy, but needs to be saturated with iron to be effective.

Induced magnetic field gradients and forces in the human head in MRI

To map the induced magnetic field gradients and estimate the magnetic force in the human head during magnetic resonance imaging at 4 Tesla (T). The magnetic field distribution in the human head was measured using two gradient-echo experiments with different echo times. The phase of the complex image ratio removed the wrapping artifact, characteristic of phase images, and was used to map the magnetic field distribution and calculate the accurate maps of the magnetic field gradients in the human head. The time-independent gradient fields induced by air/tissue interfaces in the head can be 50 times larger than those resulting from the magnetic field inhomogeneity of the MRI magnet. However, the associated magnetic force in the brain is by far smaller than the gravitational force. The induced gradient fields increase the magnetic force on tissues. However, even for tissue components with large magnetic susceptibility such as iron-containing proteins, this force is negligible compared with the gravitational force. Therefore, this study suggests that static and uniform magnetic fields do not have a significant risk for the tissues in the head.

Impact of the small RNA RyhB on growth, physiology and heterologous protein expression inEscherichia coli

The small noncoding RNA RyhB is a regulator of iron homeostasis in Escherichia coli. During iron limitation, it downregulates the expression of a number of iron-containing proteins, including enzymes of the tricarboxylic acid cycle and the respiratory chain. Because this infers a potential for RyhB to limit energy metabolism and biosynthetic capacity, the effect of knocking out ryhB on the physiology and heterologous protein productivity of E. coli has been analyzed. During iron limitation, induced either through insufficient extracellular supply or through overexpression of an iron-containing protein, ryhB mutants showed unaltered growth and substrate consumption. They did, however, exhibit significantly lowered acetate production rates. Plasmid-based expression of green fluorescent protein and the heterologous Vitreoscilla hemoglobin VHb was negatively affected by the ryhB knock-out.

Key role of the anchoring PEI layer on the electrochemistry of redox proteins at carbon electrodes

Abstract The key role of positively charged anchoring films on the electroactivity of various bacterial proteins at a pyrolytic graphite electrode is discussed. Several iron-containing low redox potential proteins, either negatively charged or positively charged were considered. Using quartz crystal microgravimetry (QCM) and electrochemical techniques, it is demonstrated that electron transfer on these proteins can be inhibited or promoted essentially depending on the electrostatic interactions with the positive PEI layer. Consequences of this interaction on the electroactivity of proteins involved in assemblies with clays are discussed. In particular, although bacterial polyhemic cytochrome c 3 is able to form stable LBL assemblies with intercalated clay layers, electroactivity of the so-mounted construction is highly dependant on the structure of the anchoring layer.

Altered Collagen in Tartrate-Resistant Acid Phosphatase (TRAP)-Deficient Mice: A Role for TRAP in Bone Collagen Metabolism

Tartrate-resistant acid phosphatase (TRAP) is an iron-containing protein that is highly expressed by osteoclasts, macrophages, and dendritic cells. The enzyme is secreted by osteoclasts during bone resorption, and serum TRAP activity correlates with resorptive activity in disorders of bone metabolism. TRAP is essential for normal skeletal development. In knockout mice lacking TRAP, bone shape and modeling is altered with increased mineral density. Here, we report the effect of TRAP on the biochemical and biomechanical properties of collagen, the major protein constituting the bone matrix, using these mice. Femurs from TRAP-/- and wild-type mice were used in these studies. The biomechanical properties were investigated using a three-point bending technique. Collagen synthesis was determined by measuring cross-link content using high-performance liquid chromatography and amino acid analysis. Collagen degradation was determined by measuring matrix metalloproteinase-2 (MMP-2) activity. The rates of collagen synthesis and degradation were significantly greater in bones from TRAP-/- mice compared with wild type. At 8 weeks, there was an increase in the intermediate cross-links but no significant difference in animals aged 6 months. There was a significant increase in mature cross-links at both ages. A significant increase in MMP-2 production both pro and active was observed. A significant increase in ultimate stress and Young's modulus of elasticity was needed to fracture the bones from mice deficient in TRAP. We conclude that both synthesis as well as degradation of collagen are increased when TRAP is absent in mice at 8 weeks and 6 months of age, showing that TRAP has an important role in the metabolism of collagen.

Structural Characterization of the Ceruloplasmin: Lactoferrin Complex in Solution

Ceruloplasmin is a copper protein found in vertebrate plasma, which belongs to the family of multicopper oxidases. Like transferrin of the blood plasma, lactoferrin, the iron-containing protein of human milk, saliva, tears, seminal plasma and of neutrophilic leukocytes tightly binds two ferric ions. Human lactoferrin and ceruloplasmin have been previously shown to interact both in vivo and in vitro forming a complex. Here we describe a study of the conformation of the human lactoferrin/ceruloplasmin complex in solution using small angle X-ray scattering. Our ab initio structural analysis shows that the complex has a 1:1 stoichiometry and suggests that complex formation occurs without major conformational rearrangements of either protein. Rigid-body modeling of the mutual arrangement of proteins in the complex essentially yields two families of solutions. Final discrimination is possible when integrating in the modeling process extra information translating into structural constraints on the interaction between the two partners.

Diiron-containing metalloproteins: Developing functional models

Abstract A major objective in protein science is the design of enzymes with novel catalytic activities that are tailored to specific applications. Such enzymes may have great potential in biocatalysis and biosensor technology, such as in degradation of pollutants and biomass, and in drug and food processing. To reach this objective, investigations into the basic biochemical functioning of metalloproteins are still required. In this perspective, metalloprotein design provides a powerful approach first to contribute to a more comprehensive understanding of the way metalloproteins function in biology, with the ultimate goal of developing novel biocatalysts and sensing devices. Metalloprotein mimetics have been developed through the introduction of novel metal-binding sites into naturally occurring proteins as well as through de novo protein design. We have approached the challenge of reproducing metalloprotein active sites by using a miniaturization process. We centered our attention on iron-containing proteins, and we developed models for heme proteins and diiron–oxo proteins. In this paper we summarize the results we obtained on the design, structural, and functional properties of DFs, a family of artificial diiron proteins.

MitoNEET is an iron-containing outer mitochondrial membrane protein that regulates oxidative capacity

Members of the thiazolidinedione (TZD) class of insulin-sensitizing drugs are extensively used in the treatment of type 2 diabetes. Pioglitazone, a member of the TZD family, has been shown to bind specifically to a protein named mitoNEET [Colca JR, McDonald WG, Waldon DJ, Leone JW, Lull JM, Bannow CA, Lund ET, Mathews WR (2004) Am J Physiol 286:E252-E260]. Bioinformatic analysis reveals that mitoNEET is a member of a small family of proteins containing a domain annotated as a CDGSH-type zinc finger. Although annotated as a zinc finger protein, mitoNEET contains no zinc, but instead contains 1.6 mol of Fe per mole of protein. The conserved sequence C-X-C-X(2)-(S/T)-X(3)-P-X-C-D-G-(S/A/T)-H is a defining feature of this unique family of proteins and is likely involved in iron binding. Localization studies demonstrate that mitoNEET is an integral protein present in the outer mitochondrial membrane. An amino-terminal anchor sequence tethers the protein to the outer membrane with the CDGSH domain oriented toward the cytoplasm. Cardiac mitochondria isolated from mitoNEET-null mice demonstrate a reduced oxidative capacity, suggesting that mito- NEET is an important iron-containing protein involved in the control of maximal mitochondrial respiratory rates.

Transferrin receptor 2 is emerging as a major player in the control of iron metabolism

Abstract Our knowledge of mammalian iron metabolism has advanced dramatically over recent years. Iron is an essential element for virtually all living organisms. Its intestinal absorption and accurate cellular regulation is strictly required to ensure the coordinated synthesis of the numerous iron-containing proteins involved in key metabolic processes, while avoiding the uptake of excess iron that can lead to organ damage. A range of different proteins exist to ensure this fine control within the various tissues of the body. Among these proteins, transferrin receptor (TFR2) seems to play a key role in the regulation of iron homeostasis. Disabling mutations in TFR2 are responsible for type 3 hereditary hemochromatosis (Type 3 HH). This review describes the biological properties of this membrane receptor, with a particular emphasis paid to the structure, function and cellular localization. Although much information has been garnered on TFR2, further efforts are needed to elucidate its function in the context of the iron regulatory network.

Crystallographic evidence for dioxygen interactions with iron proteins

The interaction of dioxygen with iron plays a key role in many important biological processes, such as dioxygen transport in the bloodstream and the reduction of dioxygen by iron in respiration. However, the catalytic mechanisms employed, for example in ligand oxidation, are not fully understood at the current time despite intensive biochemical, spectroscopic and structural studies. This review outlines the structural evidence obtained by X-ray crystallographic methods for the nature of the interactions between dioxygen and the metal in iron-containing proteins. Proteins involved in iron transport or electron transfer are not included.

Separation and Determination of Iron-Containing Proteins via Liquid Chromatography−Particle Beam/Hollow Cathode−Optical Emission Spectroscopy

Presented here is a method for the quantitative determination of iron-containing metalloproteins. Four iron-containing metalloproteins (transferrin, myoglobin, hemoglobin, and cytochrome c) were separated by high-performance liquid chromatography (HPLC) and determined through particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) by the Fe (I) 371.9 nm optical emission. Parametric optimization of sample introduction, nebulization, and hollow cathode source conditions is performed for the suite of Fe-metalloproteins. Response curves for the Fe (I) emission were obtained under optimized conditions with detection limits for triplicate injections occurring on the nanogram level for iron ( approximately 24 ng) with variability of <7% RSD over the concentration range of 0.1-100 microg/mL iron in the metalloproteins. Response curves for S (I) emission yielded similar analytical characteristics. Optical emission detection of the liquid chromatography separations of the iron-containing metalloproteins demonstrates the feasibility of the PB/HC-OES system as a simple element-specific detector for liquid chromatography. The retention times of the four analytes are similar to those determined by UV absorbance (216 nm), demonstrating the ability of the PB interface to preserve the chromatographic integrity of the separation. Additionally, empirical formula calculations based on Fe (I) and S (I) emission response ratios provide a much higher level of specificity than single-element protein determination.

Control of Fur synthesis by the non-coding RNA RyhB and iron-responsive decoding

The Fe2+-dependent Fur protein serves as a negative regulator of iron uptake in bacteria. As only metallo-Fur acts as an autogeneous repressor, Fe2+scarcity would direct fur expression when continued supply is not obviously required. We show that in Escherichia coli post-transcriptional regulatory mechanisms ensure that Fur synthesis remains steady in iron limitation. Our studies revealed that fur translation is coupled to that of an upstream open reading frame (uof), translation of which is downregulated by the non-coding RNA (ncRNA) RyhB. As RyhB transcription is negatively controlled by metallo-Fur, iron depletion creates a negative feedback loop. RyhB-mediated regulation of uof-fur provides the first example for indirect translational regulation by a trans-encoded ncRNA. In addition, we present evidence for an iron-responsive decoding mechanism of the uof-fur entity. It could serve as a backup mechanism of the RyhB circuitry, and represents the first link between iron availability and synthesis of an iron-containing protein.