The ␣ subunit of E. coli RNAP plays an important role in the recognition of many promoters by binding to the A+T-rich UP element, a DNA sequence located upstream of the recognition elements for the subunit, the −35 and −10 hexamers. We examined DNA-RNAP interactions using high resolution interference and protection footprinting methods and using the minor groove-binding drug distamycin. Our results suggest that ␣ interacts with bases in the DNA minor groove and with the DNA backbone along the minor groove, but that UP element major groove surfaces do not make a significant contribution to ␣ binding. On the basis of these and previous results, we propose a model in which ␣ contacts UP element DNA through amino acid residues located in a pair of helix-hairpin-helix motifs. Furthermore, our experiments extend existing information about recognition of the core promoter by 70 by identifying functional groups in the major grooves of the −35 and −10 hexamers in which modifications interfere with RNAP binding. These studies greatly improve the resolution of our picture of the promoter-RNAP interaction.[Key Words: RNA polymerase; promoter; alpha subunit; UP element; minor groove; helix-hairpin-helix] Promoters in bacteria contain recognition sequences for RNA polymerase (RNAP, subunit composition ␣ 2 ␤␤Ј) in up to three distinct regions: the −10 and −35 elements, both recognized by the 70 subunit (for review, see Record et al. 1996;Gross et al. 1998), and the UP element, recognized by the ␣ subunit (for review, see Gourse et al. 2000). The UP element, located just upstream of the −35 hexamer, was identified as a binding site for ␣ in the rRNA promoter rrnB P1 of Escherichia coli, where it stimulates promoter activity ∼30-fold (Ross et al. 1993). UP elements have been identified in other promoters in E. coli, in other bacterial species, and in promoters transcribed by holoenzymes containing alternative factors (e.g., Moran et al. 1982;Newlands et al. 1992;Ross et al. 1993Fredrick et al. 1995Negre et al. 1997). The optimal (consensus) UP element sequence was identified by in vitro selection and contains alternating A and T tracts in two distinct subsites (proximal and distal; Estrem et al. 1998Estrem et al. , 1999. Analysis of the E. coli genome sequence suggests that UP elements, consisting of either one or two subsites with no more than two mismatches to consensus, occur in more than one-third of stable RNA promoters, but are also found in about 4% of mRNA promoters (Estrem et al. 1999). Because sequences with much lesser degrees of match to consensus can still significantly affect promoter function , the number of promoters in which UP elements play some role in transcription is likely to be much larger. DNA-␣ interactions also occur adjacent to many activator protein-binding sites, although, in these cases, ␣ interactions are often DNA sequence independent, the binding site(s) do not resemble the UP element consensus, and ␣ binding does not occur in the absence of the bound activator (Hochschild and Dove 1998;Busby and...

The search for nitric oxide cheletropic traps (NOCTs) of the 7,7,8,8-tetraalkyl-o-quinodimethane type which would have properties appropriate for monitoring the formation of nitric oride in cell cultures and in vivo by magnetic resonance techniques is described. In addition to the necessary condition that a NOCT reacts rapidly with NO to yield a persistent nitroxide radical, two additional properties were sought: (i) thermal stability at the temperature of interest (37 "C) and (ii) water solubility. To these ends, a number of 1 ,1,3,3-tetraailcyIyl-2-indanones (and a related naphthalene derivative) were synthesized and subjected to UV photolysis in solution, a procedure which generally (though not in all cases) caused the elimination of carbon monoxide and formation of the corresponding o-quinodimethane. The thermal instability of many of these compounds is due to a 1,S-sigmatropic hydrogen atom transfer which, for example, converts 7,7,8,8-tetramethyl-o-quinodimethane (1) to o-isopropyl-a-methylstyrene ( 1 4 with a half-life of only ca. 140 s at 37 "C. Several o-quinodimethanes were discovered which were, for all practical purposes, completely stable at 37 OC. The most suitable lipid-soluble NOCT discovered was 7-(2-indenyl)-7,8,8-trimethyl-o-quinodimethane (5),which is stable and reacts very rapidly with NO to form a persistent nitroxide. Various derivatives of 5 were also examined and found to be equally, or almost equally, effective NOCTs. Water solubility was explored by addition of water-solubilizing groups to the ring of 1. The carboxylic acid group, 13, was found to be particularly suitable, since the carboxylate anion 14 conferred excellent water solubility without interfering with either the nitric oxide trapping reaction or the necessary photoelimination of carbon monoxide frpjn the starting indanone. Of even greater importance, the carboxylate group had no apparent effect on the rate of the thermal 1,s-sigmatropic rearrangement; Le., the rates of decay of 14 and 1 were equal within experimental error. It is concluded that NOCTs of the o-quinodimethane class having long lifetimes and a high reactivity toward NO can now be prepared with appropriate lipophilic, hydrophilic, or amphiphilic properties. These NOCTs should prove suitable for exploratory use in biological systems.

Reaction of the tartrate-derived diol (R,R)-d, CI, a',a'-tetraphenyl-2,2-dimethyl-1,3-dioxolane-4,5-dimethanol (TADDOL) with chlorodiphenylphosphane gives a new bis(diphenylphosphany1) ligand (TADDOP). The complex 4 formed with PdCI, has been crystallized and its structure determined by X-ray diffraction (Fig. I). The complex is used for Pd-catalyzed enantioselective 1,3-diphenylaIlylations of various nucleophiles which give products with enantiomer ratios of up to 88 : 12 (Scheme 2). Crystallization procedures lead to the enantiomerically pure ( > 99 : 1) product 11 derived from dimethyl malonate. The structure of the TADDOP complex 4 is compared with those of other transition-metal complexes containing chelating bis(diphenylphosphany1) ligands (Fig. 2). A crystallographic data base search reveals that the structures of transition-metal complexes containing two Ph2P groups (superpositions in Fzg.3) fall into one of two categories: one with approximate C, symmetry and the other with C s mmetry (20 and 19 examples, resp.). A mechanistic model is proposed which correlates the conformational chirality (6 or A ) of the four Ph groups' arrangement in such complexes with the topicity of nucleophile approach on Pd-bound trans,trans-l,3-diphenylallyl groups (Scheme 3 and Table).

Gene transcription is regulated by proteins that bind specific DNA sequences and control the initiation of RNA synthesis. A major challenge is to map all of the regulatory sites in the genome and to identify the proteins that bind them. Because members of transcription factor families often exhibit similar sequence preferences, methods for determining intermolecular contacts in protein-DNA interfaces must be sensitive to even subtle structural differences. The most detailed structural views of protein-DNA interfaces have been obtained through X-ray crystallography and NMR spectroscopy, and these methods have revolutionized the understanding of the structural determinants of sequence-specific recognition. Neither crystallography nor NMR, however, is particularly well-suited to high-throughput applications such as pan-genomic elucidation of regulatory sequences; in addition, these methods yield no information on the energetic contribution of particular contacts. Here we report a straightforward, high-resolution biochemical method for mapping, at single-nucleotide resolution, DNA bases that are subject to sequence-specific contacts by regulatory proteins.

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