The core enzyme of Escherichia coli RNA polymerase acquires essential promoter recognition and transcription initiation activities by binding one of several subunits. To characterize the proximity between 70 , the major for transcription of the growth-related genes, and the core enzyme subunits (␣ 2 ␤␤), we analyzed the proteincutting patterns produced by a set of covalently tethered DNA-dependent RNA polymerase [EC 2.7.7.6] of Escherichia coli is composed of a four-protein core enzyme (subunit composition ␣ 2 ␤␤Ј) and one of several sigma subunits. The major sigma factor, 70 (product of the rpoD gene), initiates the transcription cycle by specifying promoter recognition of most genes that are expressed during exponential cell growth and by promoting DNA strand separation (1-6). 70 binds reversibly to the ␣ 2 ␤␤Ј core enzyme (7-9), but the binding site has not yet been identified. Three-dimensional structures of the 380-kDa core enzyme (Ϸ23 Å resolution) and the 450-kDa ␣ 2 ␤␤Ј 70 holoenzyme (Ϸ30 Å resolution) provide graphic information about the overall conformations of these two macromolecular complexes (10). However, the resolution is not adequate to distinguish individual subunits, boundaries between subunits, or domains within subunits.At the level of the primary sequence, highly conserved regions have been identified previously for each of the core subunits of RNA polymerase (11, 12), but it is not known whether one or more of these regions are responsible for binding 70 . at which a small reagent with cleavage activity can be tethered. This approach employs molecular cloning to prepare a set of mutant proteins, each with a unique cysteine residue at a chosen location, for conjugation to the cutting reagent. Cutting of the other subunits occurs only at sites located near this reagent in the holoenzyme structure.As shown in Fig. 1, the 613-aa sequence of 70 has regions that are associated with core enzyme binding, recognition of hexanucleotide promoter sequences at positions Ϫ10 and Ϫ35, DNA melting, and intramolecular contacts for regulation (5,23,24). Genetic analysis has implicated two conserved regions of 70 in core binding (19,(25)(26)(27)(28). Using this information, we selected seven sites of potential interest for tethering the cutting reagent FeBABE, which can cleave peptide backbones within Ϸ12 Å of its attachment site (29). With this set of mutants, sites on the core subunits that bind near the chosen 70 sites have been identified. MATERIALS AND METHODSMaterials. N-or C-terminal subunit peptides were purchased from Phoenix Pharmaceuticals (Mountain View, CA) and used for immunization of New Zealand White rabbits. Rabbit antibodies were affinity-purified on columns containing the immobilized terminal peptide antigens. Broad-range molecular weight marker proteins were from New England Biolabs. CPM (N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenylmaleimide) was purchased from Molecular Probes. QIAprep plasmid DNA purification kits were from Qiagen. Preparative electrophoresis grade aga...

Convenient methodology for preparation and conjugation of the protein-cutting iron chelate iron (S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) is given. This formulation of the reagent can be handled in a manner analogous to many other protein-labeling reagents, such as fluorescent probes or cross-linkers. By taking advantage of the recently discovered peptide hydrolysis reaction, the chelate may be tethered to a single site (e.g., a cysteine side chain) and used to map its proximity to individual peptide bonds by automated Edman sequencing of the protein fragments produced. The method is illustrated by conjugation of Fe-BABE to the carboxy terminal domain (amino acid residues 234-329) of the Escherichia coli RNA polymerase alpha subunit. The molecular mass of the protein conjugate was confirmed by electrospray ionization mass spectrometry.

Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase alpha subunit were studied using a set of four mutant alpha subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269). These mutant alpha subunits were conjugated with the cutting reagent iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (Fe-BABE), and the peptide backbone was cleaved at locations near the modified Cys. Analysis of the cleavage sites identified segments within approximately 12 A of the conjugation site. These results show that, for intermolecular cutting, segments of the subunit assembly domain (N-terminal domain) of one subunit and the linker region between N- and C-terminal domains of the other subunit are near each other, and the N-terminal domains of both subunits are in close proximity to one another. Intramolecular cutting however, was observed only within an individual N- or C-terminal domain.

No abstract

The objective of this study was to assess the effect of dietary fibers on the transport of losartan, an angiotensin II type 1 receptor blocker, in small intestinal cells. Using Caco-2 cells in vitro, losartan uptake and transport were evaluated in the presence of various fibers (cellulose, chitosan, sodium alginate and glucomannan). Dietary fibers caused a decrease in the uptake of losartan, with chitosan causing a significant reduction. Chitosan and glucomannan significantly reduced the transport of losartan, while cellulose or sodium alginate did not. Dietary fibers also reduced the level of free losartan; however, this did not correlate with the observed reduction in losartan uptake and transport. In summary, chitosan had the greatest inhibitory effect on losartan uptake and transport, and this potential interaction should be considered in patients taking losartan. Copyright © 2016 John Wiley & Sons, Ltd.