SummaryEscherichia coli DksA, GreA, and GreB have similar structures and bind to the same location on RNA polymerase (RNAP), the secondary channel. We show that GreB can fulfill some roles of DksA in vitro, including shifting the promoter-open complex equilibrium in the dissociation direction, thus allowing rRNA promoters to respond to changes in the concentrations of ppGpp and NTPs. However, unlike deletion of the dksA gene, deletion of greB had no effect on rRNA promoters in vivo. We show that the apparent affinities of DksA and GreB for RNAP are similar, but the cellular concentration of GreB is much lower than that of DksA. When overexpressed and in the absence of competing GreA, GreB almost completely complemented the loss of dksA in control of rRNA expression, indicating its inability to regulate rRNA transcription in vivo results primarily from its low concentration. In contrast to GreB, the apparent affinity of GreA for RNAP was weaker than that of DksA, GreA only modestly affected rRNA promoters in vitro, and even when overexpressed GreA did not affect rRNA transcription in vivo. Thus, binding in the secondary channel is necessary but insufficient to explain the effect of DksA on rRNA transcription. Neither Gre factor was capable of fulfilling two other functions of DksA in transcription initiation: co-activation of amino acid biosynthetic gene promoters with ppGpp and compensation for the loss of the ω subunit of RNAP in the response of rRNA promoters to ppGpp. Our results provide important clues to the mechanisms of both negative and positive control of transcription initiation by DksA.

We show here that the promoters for many of the Escherichia coli ribosomal protein operons are regulated directly by two transcription factors, the small RNA polymerase-binding protein DksA and the nutritional stress-induced nucleotide ppGpp. ppGpp and DksA work together to inhibit transcription initiation from ribosomal protein promoters in vitro and in vivo. The degree of promoter regulation by ppGpp/DksA varies among the r-protein promoters, but some are inhibited almost as much as rRNA promoters. Thus, many r-protein operons are regulated at the level of transcription in addition to their control by the classic translational feedback systems discovered ∼30 y ago. We conclude that direct control of r-protein promoters and rRNA promoters by the same signal, ppGpp/DksA, makes a major contribution to the balanced and coordinated synthesis rates of all of the ribosomal components.ribosome synthesis | transcriptional control | stringent response | translational control P rotein synthesis is the major consumer of cellular energy in bacteria. Because the number of ribosomes is the primary determinant of the level of translation, and ribosome synthesis itself is an energy-intensive process, there are mechanisms that prevent over-or underinvestment of cellular resources in ribosome synthesis (1). Both ribosomal RNA (rRNA) and ribosomal protein (r-protein) synthesis rates are thereby tightly regulated in Escherichia coli (for reviews, see refs. 2, 3).One of the earliest reported examples of regulation of bacterial ribosome synthesis is a stress response referred to as "the stringent response," in which rRNA transcription is inhibited in cells starved for amino acids or some other nutrients (4). In this response, uncharged tRNAs induce the ribosome-associated RelA and/or SpoT proteins to synthesize ppGpp (5-7). [The term "ppGpp" is used here to describe both the unusual nucleotide guanosine-3′,5′-(bis)pyrophosphate and its pentaphosphate precursor.] ppGpp concentrations change not only after complete starvations but also after less severe shifts in nutritional conditions, coordinating rRNA synthesis with the need for protein synthesis. Shifts to a more favorable nutritional condition result in a decrease in the concentration of ppGpp and a corresponding increase in rRNA promoter activity, whereas shifts to a less favorable condition result in an increase in ppGpp and a corresponding decrease in rRNA transcription (8).ppGpp binds directly to E. coli RNA polymerase (RNAP) and inhibits transcription from rRNA promoters (9), although the identity of the ppGpp binding site on RNAP remains unclear (10). However, for ppGpp to exert its full effect on transcription, RNAP has to be modified by the small protein, DksA (11,12). Unlike ppGpp, DksA is present at high concentrations in cells under all conditions that have been examined (11,13). rRNA transcription initiation is also regulated by the concentration of the first nucleotide in the transcript (8,14) and by at least one DNA binding factor, the 11.2-kDa Fis protein (15). Tog...

Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp3 carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the hydrophobic subunits. The α,α,β,β-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be generally valuable in the development of synthetic polymers for biological applications.

SummaryThe components of the Escherichia coli flagella apparatus are synthesized in a three-level transcriptional cascade activated by the master regulator FlhDC. The cascade co-ordinates the synthesis rates of a large number of gene products with each other and with nutritional conditions. Recent genome-wide studies have reported that flagellar transcription is altered in cells lacking the transcription regulators DksA or ppGpp, but some or all reported effects could be indirect, and some are contradictory. We report here that the activities of promoters at all three levels of the cascade are much higher in strains lacking dksA, resulting in overproduction of flagellin and hyperflagellated cells. In vitro, DksA/ppGpp inhibits the flhDC promoter and the s 70 -dependent fliA promoter transcribing the gene for s 28 . However, DksA and ppGpp do not affect the s 28 -dependent fliA promoter or the s 28 -dependent fliC promoter in vitro, suggesting that the dramatic effects on expression of those genes in vivo are mediated indirectly through direct effects of DksA/ppGpp on FlhDC and s 28 expression. We conclude that DksA/ppGpp inhibits expression of the flagellar cascade during stationary phase and following starvation, thereby co-ordinating flagella and ribosome assembly and preventing expenditure of scarce energy resources on synthesis of two of the cell's largest macromolecular complexes.

bIn Escherichia coli, FadR and FabR are transcriptional regulators that control the expression of fatty acid degradation and unsaturated fatty acid synthesis genes, depending on the availability of fatty acids. In this report, we focus on the dual transcriptional regulator FadR. In the absence of fatty acids, FadR represses the transcription of fad genes required for fatty acid degradation. However, FadR is also an activator, stimulating transcription of the products of the fabA and fabB genes responsible for unsaturated fatty acid synthesis. In this study, we show that FadR directly activates another fatty acid synthesis promoter, PfabH, which transcribes the fabHDG operon, indicating that FadR is a global regulator of both fatty acid degradation and fatty acid synthesis. We also demonstrate that ppGpp and its cofactor DksA, known primarily for their role in regulation of the synthesis of the translational machinery, directly inhibit transcription from the fabH promoter. ppGpp also inhibits the fadR promoter, thereby reducing transcription activation of fabH by FadR indirectly. Our study shows that both ppGpp and FadR have direct roles in the control of fatty acid promoters, linking expression in response to both translation activity and fatty acid availability.