So far, (p)ppGpp has not been found in eukaryotes. This is not definitive evidence since its detection is technically difficult. If (p)ppGpp is not present, it is possible the Mesh1 hydrolase functions to degrade some compound other than (p)ppGpp but one that shares structural similarity to (p)ppGpp. We study structure-specificity studies with purified Mesh1 proteins in search of possible candidates that could provide functions neutralized by Mesh1. Our hypothesis is that ppGpp-like synthetic products may be diverse but each is matched by the presence of a hydrolase with a catalytic specificity. We have recently found that classical animal Mesh hydrolases uniquely have an extended specificity for cleavage of the ribose 3'-pyrophosporyl group beyond guanosine tetra-, and pentaphosphate, characteristic of standard bacterial hydrolases. A small fraction (0.07) of bacterial genomes encode Mesh homolog proteins in addition to their standard hydrolase component. We wish to ask if these unusual bacterial hydrolases also display the degenerate substrate specificity of animal hydrolases and if so, ask about the basis of catalytic site specificity. In any event, the substrate specificity of the synthetase in these genomes will be examined to see it generates products specifically by a matched hydrolase. Our long interest is to define regulation by ppGpp that works through gene-specific positive and negative regulatory effects on transcription. We have long focused on regulatory interactions between RNAP 2o channel binding proteins and ppGpp. Our analyses reveal that DksA and GreA/B can show similar and even redundant functions despite their intrinsic differences. One example is that GreA overproduction can override the amino acid requirements of a dksA mutant for growth in minimal medium (ILTV). These requirements are a subset of ppGpp0 requirements (DEFHILSTV). Another example is that GreA or DksA overexpression can reverse some of the amino acid requirements shown by ppGpp0 cells. We used microarray transcription profiles to extend conclusions of shared functions by GreA and DksA to the transcriptome. In addition to examples of redundant functions, we also find instances where GreA and DksA can act in opposition. An example of opposite effects at the phenotypic level is that complete reversal of the amino amino acid requirements of ppGpp0 cells by overexpression of GreA occurs only in the absence of DksA and vice versa. As judged by transcription profiling, more genes (331) are affected by GreA overproduction when DksA is absent than when DksA is present (45). Profiling also provides evidence that the strongest activation by GreA occurs for gadA and gadE genes. We could verify this with PgadA- and PgadE-lacZ promoter transcriptional fusions as reporters. These fusions localize activation to the level of transcription initiation rather than elongation. We also used mutants of GreA and DksA with altered key acidic residues in the tip of the coil-coil finger;these mutants abolish their classical functions but do not alter the regulation of selected promoter-lacZ fusions. This strongly suggests that the regulatory features we have uncovered represent a new phenomenon. They are not simple variations of the usual activities of these secondary channel proteins. These results reinforce our earlier notions that a complex interplay between GreA, GreB, and DksA exists in the absence of ppGpp. Mechanistically, this could occur by promoter activation, competition for RNA polymerase at the level of either binding, secondary channel occupancy, or both, not to mention mutual control of one another's gene expression. Our transcriptional profiling study serves to localize some of these interactions to specific genes and regulators, which might show promise for uncovering new regulatory mechanisms at the molecular level. We also explored the interactions between these three factors in a hierarchical series of RNAP mutants of increasing severity that allowed ppGpp0 strains to grow without amino acids. The work confirms that the ppGpp-dependence of factor interactions operates at the level RNAP. Overall, these experiments reveal that a much more complex and generalized regulatory interplay exists between GreA, GreB and DksA than was previously appreciated.

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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
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Potrykus, Katarzyna; Murphy, Helen; Chen, Xiongfong et al. (2010) Imprecise transcription termination within Escherichia coli greA leader gives rise to an array of short transcripts, GraL. Nucleic Acids Res 38:1636-51
Blankschien, Matthew D; Potrykus, Katarzyna; Grace, Elicia et al. (2009) TraR, a homolog of a RNAP secondary channel interactor, modulates transcription. PLoS Genet 5:e1000345
Rhee, Hyun-Woo; Lee, Chang-Ro; Cho, Seung-Hyon et al. (2008) Selective fluorescent chemosensor for the bacterial alarmone (p)ppGpp. J Am Chem Soc 130:784-5
Harinarayanan, Rajendran; Murphy, Helen; Cashel, Michael (2008) Synthetic growth phenotypes of Escherichia coli lacking ppGpp and transketolase A (tktA) are due to ppGpp-mediated transcriptional regulation of tktB. Mol Microbiol 69:882-94

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