ppGpp affects carbohydrate-dependent growth transitions, but differently than cAMP. When cells grow exponentially on mixtures of glucose and lactose, they first exhaust the glucose, then growth lags transiently because adjustments of gene expression are needed before growth can resume on lactose. This growth pause between serial use of two sugars is termed a diauxic lag. Early cAMP discoveries revealed it represses the utilization of lactose until glucose is exhausted and subsequently found that AMP interacts a protein bound to lac operon promoter DNA to turn off of lactose in preference to glucose. The abundance of (p)ppGpp is known to affect this process. Levels of ppGpp are determined by the balance of activities of two synthetases and one hydrolase. We compared diauxic lags of wild type with mutants of one or another ppGpp synthetase under many conditions, including controls that bypass cAMP effects and directly measured (p)ppGpp levels. These efforts suggest a surprising explanation. Acetate is measurably formed and excreted during growth on glucose, only to later be reabsorbed and, in the presence of (p)ppGpp, be converted to acetyphosphate (Ac-P). This pathway is the reverse of the normal pathway for making Ac-P from acetyl-CoA. Ac-P is a high energy donor for post-translational acetylation of a large number of proteins that alter activities of glycolysis and the TCA cycle. Enhanced protein acetylation was confirmed during the lag by Westerns with Anti-acetyl-lysine monoclonal antibody. Finally, we found that basal ppGpp levels are correlate directly with basal Ac-P levels. While details of the mechanism underlying this correlation remain to be uncovered, this reveals a new level of regulatory complexity: global transcriptional regulation by ppGpp appears to intersect with its regulation of a known global posttranslational regulator, Ac-P. Phage T4 Transcription during infection is reduced without dksA but not without ppGpp. Dr. Tamara James observed as a postdoc a few years ago that the plaque sizes of phage T4 infecting wild type hosts were larger with infected E. coli deleted for dksA or ppGpp (ppGppo). This is of interest since ppGpp and DksA often act directly on RNA polymerase. The 20 minute time course of the T4 infection cycle consists of early, middle and late transcripts. Early promoters use unmodified RNA polymerase and early transcripts do not terminate but instead read through middle promoters. Middle transcripts are made with a modified RNAP that recruits early proteins, MotA and AsiA. Late promoters use a RNAP additionally modified by three middle proteins. Biological measures during infection included a new semi-automated statistical method devised to document plaque size populations along with classical estimates of burst sizes and latent periods. The NICHD Sequencing Core performed RNA-seq analysis complemented by our RT-qPCR and primer extension analyses. The effect of dksA is to double burst size without changing the timing of phage release and to increase early and some middle transcripts by readthrough. The increased middle gene transcripts appear to partially compensate for RNAP modification defects for mutant phage with a partly active MotA protein. In vitro assays of early promoter transcripts with RNA polymerase revealed DksA did not inhibit activity suggesting effects of deletions in vivo are indirect. Surprisingly, ppGpp0 had only modest effects on all parameters measured despite statistical documentation of enlarged plaques. Direct ppGpp assays might address the possibility that infection quickly abolishes the ppGpp accumulation response. This work has already attracted attention since it may provide a way to enhance the yield of surface vectors loaded with therapeutic payloads with cell-specific targets. This is the first study of effects of DksA and ppGpp on lytic bacterial phage development. Patterson-West J, James TD, Fernndez-Coll L, Iben JR, Moon K, Knipling L, Cashel M, Hinton DM. 2018. Viruses. 6:10(6). doi: 10.3390/v10060308 PMID: 29882792. ppGpp alters RNAP structure to enhance DksA inhibition of rrnB P1 promoter initiation. A key element for triggering the global stringent regulatory response is inhibition of ribosomal RNA synthesis at ribosomal promoters. Two distinct ppGpp binding sites on RNAP sites are known from our work and from others. Site 1 is located on a boundary of the large subunit and the smallest subunit; omega mutants are viable with modest effects on the stringent response. Site 2 is 60 A away from site 1 and formed only in the presence of DksA binding; DksA adversely alters rrnB P1 inhibition and the stringent response. In collaboration with the Murakami laboratory, preformed RNAP holoenzyme crystals were soaked with DksA alone or with the added presence of ppGpp. This resulted in structural documentation of conformational changes due to each addition. Bound DksA has access to both the template strand at the active site and a downstream DNA site. Addition of ppGpp modifies this complex in a manner that explains its ability to inhibit rrnB P1 initiation by collapsing open complexes before they can initiate RNA synthesis and form the first diester bond. Our laboratory has shown cellular ppGpp is a more potent inhibitor than pppGpp and that site 1 binds both ppGpp and pppGpp. Similar studies might reveal whether different affinities of the two isomers for site 2 might account for this behavior. These observations provide definitive structural evidence for allosteric effects of ppGpp and DksA on transcription regulation. Opposing effects of (p)ppApp and (p)ppGpp on RNA polymerase structure and function. As mentioned in earlier annual reports, Dr. K. Potrykus, while a postdoc our lab, discovered a substrate specificity difference between a eukaryotic (p)ppGpp Mesh hydrolase and the E.coli and Streptococcal equisimilis hydrolases. The bacterial hydrolases cleave (p)ppGpp but not (p)ppApp while the Mesh enzyme cleaves both. The most recent step in our continuing collaboration with the Potrykus lab compared (p)ppApp isomers and (p)ppGpp isomers with respect to effects on the standard rrnB P1 promoter activity and, in collaboration with the Murakami laboratory, also identified a new RNA polymerase binding site. The adenine analogs were demonstrated to activate the standard rrnB P1 promoter transcripts. Regulatory features of the adenine analogs on transcription were assessed and found to be more modest than those of (p)pGpp. Surprisingly, they often were the opposite of those known for (p)ppGpp. Three examples are: i- (p)ppApp activates initiation of transcription whereas (p)ppGpp inhibits; ii- pppApp is a more potent activator than ppApp whereas ppGpp is more potent inhibitor than pppGpp; and iii- pppApp activation of transcription stabilizes open complexes whereas ppGpp destabilizes open complexes. Evidently, a yin-yang regulatory relationship exists between pppApp and ppGpp. Order of addition experiments reveal that the analog added first dominates the regulatory outcome over the regulatory effect of the one added later and vice versa. This suggests different binding sites for the G and A analogs as well as a high degree of plasticity of RNAP conformational changes, seemingly each kinetically stable enough that once formed are able to persist despite the added presence of the second analog. Soaking pppApp into RNA holoenzyme crystals reveals that pppApp binds to an entirely new site located near the catalytic polymerization site near the switch region. The new site is distinct from the DksA-ppGpp binding site 2 and distinct from ppGpp binding site 1. This comparison of (p)ppApp and (p)ppGpp analogs suggests that during evolution RNA polymerase might well have evolved to harbor a (p)ppApp-specific binding site, which could imply a widespread biological occurrence.
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