SUMMARY NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a β-barrel in NusG and an α-hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here we show that the released RfaH-CTD completely refolds from an all-α to an all-β conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated.

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.

One of the principal peptide components of the amyloid plaque deposits of Alzheimer's disease in humans is the 40-amino-acid peptide 8-amyloid A4-( 1 -40)-peptide. The full-length A4-( I -40)-peptide was chemically synthesized and the solution structure determined by two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular-dynamics calculations. Synthetic human A4-( 1 -40)-peptide was soluble and non-aggregating for several days in 40 % (by vol.) trifluoroethanol/water. All spin systems could be unambiguously assigned, and a total of 203 sequential and medium-range crosspeaks were found in the NOESY (nuclear Overhauser enhancement spectroscopy) spectrum. Long-range NOE cross-peaks that would indicate tertiary structure of the peptide were absent. The main secondarystructure elements found by chemical-shift analysis, sequential and medium-range NOESY data, and NOE-based restrained molecular-dynamics calculations were two helices, Glnl5 -Asp23 and Ile31-Met35, whereas the rest of the peptide was in random-coil conformation. A similar secondary structure is suggested for the aggregation part of prions, the postulated causative agents of the transmissible spongiform encephalopathy. The sequence of the helical part of prion proteins was observed to be remarkably similar to the sequence of the helical part of human A4-(1-40)-peptide.

NusG is a conserved regulatory protein that interacts with elongating complexes (ECs) of RNA polymerase (RNAP), DNA, and RNA to modulate transcription in multiple and sometimes opposite ways. In E. coli, NusG suppresses pausing and increases elongation rate, enhances termination by E. coli ρ and phage HK022 Nun protein, and promotes antitermination by λN and in ribosomal RNA operons. We report NMR studies that suggest E. coli NusG consists of two largely independent Nand C-terminal structural domains, NTD and CTD. 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 ρ-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 RNAP and the CTD interacts with ρ and other regulators, providing NusG with different combinations of interactions to effect different regulatory outcomes.

ObjectiveIt is well known that total cholesterol becomes less of a risk factor or not at all for all-cause and cardiovascular (CV) mortality with increasing age, but as little is known as to whether low-density lipoprotein cholesterol (LDL-C), one component of total cholesterol, is associated with mortality in the elderly, we decided to investigate this issue.Setting, participants and outcome measuresWe sought PubMed for cohort studies, where LDL-C had been investigated as a risk factor for all-cause and/or CV mortality in individuals ≥60 years from the general population.ResultsWe identified 19 cohort studies including 30 cohorts with a total of 68 094 elderly people, where all-cause mortality was recorded in 28 cohorts and CV mortality in 9 cohorts. Inverse association between all-cause mortality and LDL-C was seen in 16 cohorts (in 14 with statistical significance) representing 92% of the number of participants, where this association was recorded. In the rest, no association was found. In two cohorts, CV mortality was highest in the lowest LDL-C quartile and with statistical significance; in seven cohorts, no association was found.ConclusionsHigh LDL-C is inversely associated with mortality in most people over 60 years. This finding is inconsistent with the cholesterol hypothesis (ie, that cholesterol, particularly LDL-C, is inherently atherogenic). Since elderly people with high LDL-C live as long or longer than those with low LDL-C, our analysis provides reason to question the validity of the cholesterol hypothesis. Moreover, our study provides the rationale for a re-evaluation of guidelines recommending pharmacological reduction of LDL-C in the elderly as a component of cardiovascular disease prevention strategies.

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.

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.Antibiotics are chemicals that are active against microorganisms, exerting their function in different ways at various cellular locations. Aminoglycoside antibiotics, for example, target the 30 S subunit of ribosomal RNA and cause mistranslation. Molecules of the neomycin family of aminoglycosides ( Fig. 1a) bind directly to the A site of 16 S ribosomal RNA (1) and efficiently disturb protein biosynthesis of prokaryotes. Structural studies on the interaction of aminoglycosides with RNAs provided insights into the mechanisms of miscoding (2-4). The antibiotic distorts the structure of the RNA and thus leads to errors in protein biosynthesis. Variations in eukaryotic ribosomal RNA prevent high affinity binding of aminoglycosides to the ribosomes of higher organisms, making them less prone to antibiotic influence and thus rendering the antibiotics valuable medical drugs. Due to the growing problem of antibiotic resistance, caused by only a small number of mutations in the microorganisms, the determinants for antibiotic binding to RNA are of major interest in structural biology. Only a few structures of antibiotic-RNA complexes have been determined experimentally to date, among them the structure of paromomycin in complex with a model oligonucleotide comprising the A site of 16 S rRNA (2) and a low resolution and two high resolution structures of complexes between RNA aptamers and tobramycin or neomycin (5-7).Aminoglycosides have also been found to bind to group I introns (8), to hammerhead RNA (9), and to human hepatitis ␦ virus ribozymes (10). These antibiotics also bind to the Rev (regulator of expression of the virion) and Tat (transactivator of transcription) binding regions of human immunodeficiency virus type 1 (HIV-1) 1 RNA, Rev response element (11), and Tat-responsive element (TAR) (12). Different modeling ap...

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