1-Substituted diazen-1-ium-1,2-diolates have proved useful as tools in enzymology and other pharmacological research applications in which spontaneous generation of nitric oxide according to a reasonably well-defined time course is required This chapter summarizes relevant physicochemical data, including the NO release rates and product profiles, for a selection of these compounds Guidelines for quality control and a systematic nomenclature scheme are also presented It is hoped that, in summarizing this information here, our chapter will help those contemplating new applications of diazeniumdiolate technology as they seek to capitalize on what we believe are the inherent advantages of this compound type in research on the pharmacological properties of nitric oxide

"NONOates" (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors: convenient nitric oxide dosage forms

The UP element, a component of bacterial promoters located upstream of the −35 hexamer, increases transcription by interacting with the RNA polymerase α-subunit. By using a modification of the SELEX procedure for identification of protein-binding sites, we selected in vitro and subsequently screened in vivo for sequences that greatly increased promoter activity when situated upstream of the Escherichia coli rrnB P1 core promoter. A set of 31 of these upstream sequences increased transcription from 136- to 326-fold in vivo, considerably more than the natural rrnB P1 UP element, and was used to derive a consensus sequence: −59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn −38. The most active selected sequence contained the derived consensus, displayed all of the properties of an UP element, and the interaction of this sequence with the α C-terminal domain was similar to that of previously characterized UP elements. The identification of the UP element consensus should facilitate a detailed understanding of the α–DNA interaction. Based on the evolutionary conservation of the residues in α responsible for interaction with UP elements, we suggest that the UP element consensus sequence should be applicable throughout eubacteria, should generally facilitate promoter prediction, and may be of use for biotechnological applications.

https://www.pnas.org/content/pnas/95/17/9761.full.pdf

Identification of an UP element consensus sequence for bacterial promoters

The chemistry of nitroxyl (HNO) and implications in biology

To face physicochemical and biological stresses, living organisms evolved endogenous chemical responses based on gas exchange with the atmosphere and on formation of nitric oxide (NO(*)) and oxygen derivatives. The combination of these species generates a complex network of variable extension in space and time, characterized by the nature and level of the reactive oxygen (ROS) and nitrogen species (RNS) and of their organic and inorganic scavengers. Among the latter, this review focusses on natural 3-substituted indolic structures. Tryptophan-derived indoles are unsensitive to NO(*), oxygen and superoxide anion (O(2)(*-)), but react directly with other ROS/RNS giving various derivatives, most of which have been characterized. Though the detection of some products like kynurenine and nitroderivatives can be performed in vitro and in vivo, it is more difficult for others, e.g., 1-nitroso-indolic compounds. In vitro chemical studies only reveal the strong likelihood of their in vivo generation and biological effects can be a sign of their transient formation. Knowing that 1-nitrosoindoles are NO donors and nitrosating agents indicating they can thus act both as mutagens and protectors, the necessity for a thorough evaluation of indole-containing drugs in accordance with the level of the oxidative stress in a given pathology is highlighted.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1600-079X.2008.00580.x

Potential role of tryptophan derivatives in stress responses characterized by the generation of reactive oxygen and nitrogen species

Promoter sequences involved in recognition by Escherichia coli RNA polymerase (RNAP) were identified from comparisons of a large number of known promoters and from mutational analyses (28, 29, 38, 62). These sequences, the −10 and −35 hexamers (5′ TATAAT 3′ and 5′TTGACA 3′, respectively), are recognized by the ς70 subunit of RNAP (11). The strength of a promoter correlates generally with its degree of identity to these sequences and with the length of the spacer between them (the homology score [42]), although exceptions to this rule have been described (7, 26).

It was proposed more than 10 years ago that optimal transcription activity could be achieved by different combinations of promoter elements, including not only the −10 and −35 hexamers, but also upstream and downstream regions (7). In accord with this suggestion, RNAP protects regions both upstream and downstream of the −10 and −35 hexamers in footprints (8, 45, 47, 56), and A+T-rich sequences upstream of the −35 hexamer in several E. coli or Bacillus subtilis promoters were found to increase transcription in vitro in the absence of accessory proteins (3, 19, 31, 37, 39, 50, 54). Phased A-tracts inserted upstream of the −35 region in various promoter constructs were also shown to increase transcription (6, 12, 24).

The A+T-rich region upstream of −40 in the rRNA promoter rrnB P1, the UP element, increases transcription 30- to 70-fold by binding the RNAP α subunit (13, 50, 53). A consensus UP element sequence was determined by using in vitro selection for upstream sequences that promote rapid RNAP binding to the rrnB P1 promoter, followed by in vivo screening for high promoter activity. The consensus UP element consists of alternating A- and T-tracts (13). UP elements matching the consensus increased promoter activity as much as 326-fold, about 5-fold more than the wild-type rrnB P1 UP element. UP elements were also identified in other promoters, for example, the flagellin (hag) promoter of B. subtilis (18), the PL2 promoter of phage lambda (25), and the Pe promoter of phage Mu (61), although the effects of these elements were not as large as that of rrnB P1. UP elements also function in promoters recognized by RNAP holoenzymes with alternate ς factors (18).

UP elements are not as highly conserved as the −10 and −35 elements and were not described in studies comparing the large sets of E. coli promoters used to define the consensus hexamers (28, 29, 38). However, A+T-rich sequences were identified as a prominent feature of a subset of E. coli promoters (the −44 motif [23]), and a recent E. coli promoter analysis (48) identified two A+T-rich regions at upstream positions corresponding to those crucial for UP element function (14). A+T-rich upstream sequences were also identified in compilations of B. subtilis and Clostridium promoters (27, 30).

We have proposed that UP elements may be a recognition feature in many bacterial promoters (13, 53), but in most promoters, the role of upstream sequences has not been evaluated experimentally. Therefore, in this paper, we have examined the role of upstream sequences from six promoters (rrnB P2, rrnD P1, RNA II, merT, lac, and λ pR). We find that several of the sequences function as UP elements and that their effects on promoter activity differ, correlating generally with the degree of similarity to the UP element consensus sequence. These results support the model that bacterial promoters consist of at least three modules, not just −10 and −35 elements. We also show that upstream protection in footprints is not a reliable indicator of UP element function.

L.K. Keefer

Papers

Experimental Tests of the Mutagenicity and Carcinogenicity of Nitric Oxide and Its Progenitors