We continue to study a large number of both general and specific effects of ppGpp that relate to the integration of molecular regulation of bacterial physiology and gene expression. We have four reviews this year of these topics. One of the major surprises of our ongoing work is repeatedly finding that the common views of specificity of transcription factor functions are somewhat blurred. For example, the GreA transcription factor that classically affects elongation has been described in our earlier reports to also alter open complex formation during initiation of ribosomal promoters that are inhibited by ppGpp. In special genetic backgrounds, GreA overproduction can also reverse requirements for several amino acids (ILVSTFDQH) biosynthetic phenotypes occasioned by a ppGpp-deficiency. This year (Nucleic Acids Research reference), we report that greA transcripts are unusual because of the existence of a special terminator between two tandem greA promoters and the greA ORF. Because of this terminator, about 2/3ds of the total transcripts terminate imprecisely over a 10 nt range. The two promoters generate two small RNA chain populations with lengths of 40-50 nt and 50-60nt. This is the first discovery of this degree of imprecise termination for E. coli for an """"""""intrinsic"""""""" terminator, (one that functions without additional factors). The possibility that these small RNA populations have regulatory functions is underscored by fitness assays, by evolutionary conservation and by transcriptional profiling. This year we also report our re-evaluation of the role of ppGpp as a determinant of enteric bacteria cellular content of DNA, RNA and protein as a function of growth rate (Environmental Microbiology reference). It has been known for over 50 years that the cellular content of these macromolecules was controlled solely by the exponential growth rate and not the composition of the growth media needed to achieve a given growth rate. This regulatory phenomenon is called growth rate control. Since our discovery over 40 years ago of ppGpp and that ppGpp could negatively regulate rRNA synthesis during the stress of amino acid starvation, it has been uncertain whether ppGpp causes growth rate control at all, in part, or fully. During nearly the past two decades the controversy has been limited to the question of whether or not multiple redundant regulatory systems exist in addition to ppGpp. These past discordant results have arisen in other laboratories using strains we constructed to be completely deficient in ppGpp. Using similar strains, our re-evaluation differs from earlier experiments through the use of especially sensitive assay techniques (fluorescent dyes), development of nutritionally defined media for growing ppGpp-deficient cells for the first time and deriving special culture conditions to avoid suppressor mutations in RNA polymerase that otherwise confound results. We believe our findings apparently finally resolve this seemingly intractable controversy. Our conclusion also greatly simplifies the explanation for growth rate control in E. coli because ppGpp is found to be the major source of growth rate control.

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Molodtsov, Vadim; Sineva, Elena; Zhang, Lu et al. (2018) Allosteric Effector ppGpp Potentiates the Inhibition of Transcript Initiation by DksA. Mol Cell 69:828-839.e5
Potrykus, Katarzyna; Cashel, Michael (2018) Growth at best and worst of times. Nat Microbiol 3:862-863
Fernández-Coll, Llorenç; Cashel, Michael (2018) Contributions of SpoT Hydrolase, SpoT Synthetase, and RelA Synthetase to Carbon Source Diauxic Growth Transitions in Escherichia coli. Front Microbiol 9:1802
Bruhn-Olszewska, Bo?ena; Molodtsov, Vadim; Sobala, Micha? et al. (2018) Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro. Biochim Biophys Acta Gene Regul Mech 1861:731-742
Kamarthapu, Venu; Epshtein, Vitaly; Benjamin, Bradley et al. (2016) ppGpp couples transcription to DNA repair in E. coli. Science 352:993-6
Gaca, Anthony O; Kudrin, Pavel; Colomer-Winter, Cristina et al. (2015) From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis. J Bacteriol 197:2908-19
Mechold, Undine; Potrykus, Katarzyna; Murphy, Helen et al. (2013) Differential regulation by ppGpp versus pppGpp in Escherichia coli. Nucleic Acids Res 41:6175-89
Vinella, Daniel; Potrykus, Katarzyna; Murphy, Helen et al. (2012) Effects on growth by changes of the balance between GreA, GreB, and DksA suggest mutual competition and functional redundancy in Escherichia coli. J Bacteriol 194:261-73
Edwards, Adrianne N; Patterson-Fortin, Laura M; Vakulskas, Christopher A et al. (2011) Circuitry linking the Csr and stringent response global regulatory systems. Mol Microbiol 80:1561-80
James, Tamara D; Cashel, Michael; Hinton, Deborah M (2010) A mutation within the {beta} subunit of Escherichia coli RNA polymerase impairs transcription from bacteriophage T4 middle promoters. J Bacteriol :

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