Paul Rösch

A NusE:NusG Complex Links Transcription and Translation

Transcription and Translation in Train In bacteria, translation of messenger RNA into proteins by the ribosome usually begins soon after the ribosome binding site emerges from RNA polymerase. Now there is evidence for direct coupling between transcription and translation in bacteria. Proshkin et al. (p. 504; see the Perspective by Roberts) show that the trailing ribosome controls the rate of transcription by preventing RNA polymerase from spontaneous backtracking, which allows precise adjustment of transcriptional yield to translational needs under various growth conditions. Burmann et al. (p. 501; see the Perspective by Roberts) provide a potential mechanism for coupling by showing that the transcription factor NusG, which binds RNA polymerase through its amino-terminal domain, competitively binds either a ribosomal protein or the Rho transcription termination factor through its carboxy-terminal domain. Rho binding might occur after release of the ribosome from messenger RNA, thus linking termination of transcription and translation. The ribosome pushes RNA polymerase to prevent backtracking, which links rates of translation with transcription. Bacterial NusG is a highly conserved transcription factor that is required for most Rho activity in vivo. We show by nuclear magnetic resonance spectroscopy that Escherichia coli NusG carboxyl-terminal domain forms a complex alternatively with Rho or with transcription factor NusE, a protein identical to 30S ribosomal protein S10. Because NusG amino-terminal domain contacts RNA polymerase and the NusG carboxy-terminal domain interaction site of NusE is accessible in the ribosomal 30S subunit, NusG may act as a link between transcription and translation. Uncoupling of transcription and translation at the ends of bacterial operons enables transcription termination by Rho factor, and competition between ribosomal NusE and Rho for NusG helps to explain why Rho cannot terminate translated transcripts.

Structural studies of HIV-1 tat protein

Tat (trans-activator) proteins are early RNA binding proteins regulating lentiviral transcription. These proteins are necessary components in the life cycle of all known lentiviruses, such as the human immunodeficiency viruses (HIV) or the equine infectious anemia virus (EIAV). Tat proteins are thus ideal targets for drugs intervening with lentiviral growth. The consensus RNA binding motif (TAR, trans-activation responsive element) of HIV-1 is well characterized. Structural features of the 86 amino acid HIV-1, Zaire 2 isolate (HV1Z2) Tat protein in solution were determined by two dimensional (2D) nuclear magnetic resonance (NMR) methods and molecular dynamics (MD) calculations. In general, sequence regions corresponded to structural domains of the protein. It exhibited a hydrophobic core of 16 amino acids and a glutamine-rich domain of 17 amino acids. Part of the NH2 terminus, Val4 to Pro14, was sandwiched between these domains. Two highly flexible domains corresponded to a cysteine-rich and a basic sequence region. The 16 amino acid sequence of the core region is strictly conserved among the known Tat proteins, and the three-dimensional fold of these amino acids of HV1Z2 Tat protein was highly similar to the structure of the corresponding EIAV Tat domain. HV1Z2 Tat protein contained a well defined COOH-terminal Arg-Gly-Asp (RGD) loop similar to the recently determined decorsin RGD loop.

Structure and stability of 2S albumin-type peanut allergens: implications for the severity of peanut allergic reactions.

Resistance to proteolytic enzymes and heat is thought to be a prerequisite property of food allergens. Allergens from peanut (Arachis hypogaea) are the most frequent cause of fatal food allergic reactions. The allergenic 2S albumin Ara h 2 and the homologous minor allergen Ara h 6 were studied at the molecular level with regard to allergenic potency of native and protease-treated allergen. A high-resolution solution structure of the protease-resistant core of Ara h 6 was determined by NMR spectroscopy, and homology modelling was applied to generate an Ara h 2 structure. Ara h 2 appeared to be the more potent allergen, even though the two peanut allergens share substantial cross-reactivity. Both allergens contain cores that are highly resistant to proteolytic digestion and to temperatures of up to 100 degrees C. Even though IgE antibody-binding capacity was reduced by protease treatment, the mediator release from a functional equivalent of a mast cell or basophil, the humanized RBL (rat basophilic leukaemia) cell, demonstrated that this reduction in IgE antibody-binding capacity does not necessarily translate into reduced allergenic potency. Native Ara h 2 and Ara h 6 have virtually identical allergenic potency as compared with the allergens that were treated with digestive enzymes. The folds of the allergenic cores are virtually identical with each other and with the fold of the corresponding regions in the undigested proteins. The extreme immunological stability of the core structures of Ara h 2 and Ara h 6 provides an explanation for the persistence of the allergenic potency even after food processing.

Structure Determination of Human and Murine Beta-Defensins Reveals Structural Conservation in the Absence of Significant Sequence Similarity

Defensins are cationic and cysteine‐rich peptides that play a crucial role in the host defense against microorganisms of many organisms by their capability to permeabilize bacterial membranes. The low sequence similarity among the members of the large mammalian β‐defensin family suggests that their antimicrobial activity is largely independent of their primary structure. To investigate to what extent these defensins share a similar fold, the structures of the two human β‐defensins, hBD‐1 and hBD‐2, as well as those of two novel murine defensins, termed mBD‐7 and mBD‐8, were determined by nuclear magnetic resonance spectroscopy. All four defensins investigated share a striking similarity on the level of secondary and tertiary structure including the lack of a distinct hydrophobic core, suggesting that the fold is mainly stabilized by the presence of three disulfide bonds. In addition to the overall shape of the molecules, the ratio of solvent‐exposed polar and hydrophobic side chains is also very similar among the four defensins investigated. It is significant that β‐defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the β‐defensin‐specific fold appears to accommodate a wide range of different amino acids at most sequence positions. In addition to the implications for the mode of biological defensin actions, these findings are of particular interest because β‐defensins have been suggested as lead compounds for the development of novel peptide antibiotics for the therapy of infectious diseases.

Two Structurally Independent Domains of E. coli NusG Create Regulatory Plasticity via Distinct Interactions with RNA Polymerase and Regulators

NusG is a conserved regulatory protein that interacts with elongation complexes (ECs) of RNA polymerase, DNA, and RNA to modulate transcription in multiple and sometimes opposite ways. In Escherichia coli, NusG suppresses pausing and increases elongation rate, enhances termination by E. coli rho and phage HK022 Nun protein, and promotes antitermination by lambdaN and in ribosomal RNA operons. We report NMR studies that suggest that E. coli NusG consists of two largely independent N- and C-terminal structural domains, NTD and CTD, respectively. Based on tests of the functions of the NTD and CTD and variants of NusG in vivo and in vitro, we find that NTD alone is sufficient to suppress pausing and enhance transcript elongation in vitro. However, neither domain alone can enhance rho-dependent termination or support antitermination, indicating that interactions of both domains with ECs are required for these processes. We propose that the two domains of NusG mediate distinct interactions with ECs: the NTD interacts with RNA polymerase and the CTD interacts with rho and other regulators, providing NusG with different combinations of interactions to effect different regulatory outcomes.

The structure of iron–sulfur proteins

Ferredoxins are a group of iron-sulfur proteins for which a wealth of structural and mutational data have recently become available. Previously unknown structures of ferredoxins which are adapted to halophilic, acidophilic or hyperthermophilic environments and new cysteine patterns for cluster ligation and non-cysteine cluster ligation have been described. Site-directed mutagenesis experiments have given insight into factors that influence the geometry, stability, redox potential, electronic properties and electron-transfer reactivity of iron-sulfur clusters.

Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B.

Binding of human immunodeficiency virus type 1 (HIV-1) transactivator (Tat) protein to Tat-responsive RNA (TAR) is essential for viral replication and is considered a promising starting point for the design of anti-HIV drugs. NMR spectroscopy indicated that the aminoglycosides neomycin B and ribostamycin bind to TAR and that neomycin is able to inhibit Tat binding to TAR. The solution structure of the neomycin-bound TAR has been determined by NMR spectroscopy. Chemical shift mapping and intermolecular nuclear Overhauser effects define the binding region of the aminoglycosides on TAR and give strong evidence for minor groove binding. Based on 15 nuclear Overhauser effect-derived intermolecular distance restraints, a model structure of the TAR-neomycin complex was calculated. Neomycin is bound in a binding pocket formed by the minor groove of the lower stem and the uridine-rich bulge of TAR, which adopts a conformation different from those known. The neamine core of the aminoglycoside (rings I and II) is covered with the bulge, explaining the inhibition of Tat by an allosteric mechanism. Neomycin reduces the volume of the major groove in which Tat is bound and thus impedes essential protein-RNA contacts.

Mutational epitope analysis of Pru av 1 and Api g 1, the major allergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgE reactivity with three-dimensional structure.

Birch pollinosis is often accompanied by adverse reactions to food due to pollen-allergen specific IgE cross-reacting with homologous food allergens. The tertiary structure of Pru av 1, the major cherry (Prunus avium) allergen, for example, is nearly identical with Bet v 1, the major birch (Betula verrucosa) pollen allergen. In order to define cross-reactive IgE epitopes, we generated and analysed mutants of Pru av 1 and Api g 1.0101, the major celery (Apium graveolens) allergen, by immunoblotting, EAST (enzyme allergosorbent test), CD and NMR spectroscopy. The mutation of Glu45 to Trp45 in the P-loop region, a known IgE epitope of Bet v 1, significantly reduced IgE binding to Pru av 1 in a subgroup of cherry-allergic patients. The backbone conformation of Pru av 1 wild-type is conserved in the three-dimensional structure of Pru av 1 Trp45, demonstrating that the side chain of Glu45 is involved in a cross-reactive IgE epitope. Accordingly, for a subgroup of celery-allergic patients, IgE binding to the homologous celery allergen Api g 1.0101 was enhanced by the mutation of Lys44 to Glu. The almost complete loss of IgE reactivity to the Pru av 1 Pro112 mutant is due to disruption of its tertiary structure. Neither the mutation Ala112 nor deletion of the C-terminal residues 155-159 influenced IgE binding to Pru av 1. In conclusion, the structure of the P-loop partially explains the cross-reactivity pattern, and modulation of IgE-binding by site-directed mutagenesis is a promising approach to develop hypo-allergenic variants for patient-tailored specific immunotherapy.

CD and NMR Studies of Prion Protein (PrP) Helix 1 NOVEL IMPLICATIONS FOR ITS ROLE IN THE PrPC→ PrPSc CONVERSION PROCESS

The conversion of prion helix 1 from an α-helical into an extended conformation is generally assumed to be an essential step in the conversion of the cellular isoform PrPC of the prion protein to the pathogenic isoform PrPSc. Peptides encompassing helix 1 and flanking sequences were analyzed by nuclear magnetic resonance and circular dichroism. Our results indicate a remarkably high instrinsic helix propensity of the helix 1 region. In particular, these peptides retain significant helicity under a wide range of conditions, such as high salt, pH variation, and presence of organic co-solvents. As evidenced by a data base search, the pattern of charged residues present in helix 1 generally favors helical structures over alternative conformations. Because of its high stability against environmental changes, helix 1 is unlikely to be involved in the initial steps of the pathogenic conformational change. Our results implicate that interconversion of helix 1 is rather representing a barrier than a nucleus for the PrPC→ PrPSc conversion.

Should we lower cholesterol as much as possible

Statins are portrayed as harmless drugs that almost everyone would benefit from, but little is known about the side effects at the high doses now being suggested