Integration Of Macromolecular Synthesis

Child Health And Human Development

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Abstract

We wish to understand how global patterns of bacterial gene expression are coordinated by nutrient availability and stress. Research focus is on (p)ppGpp, which are two regulatory nucleotide analogs of GTP and GDP that are widespread in bacteria and plants but not in animal cells. The literature shows that (p)ppGpp compounds in different organisms can inhibit growth, promote virulence gene expression or coordinate gene expression during stress responses, thereby offering a possibility for antibiotic development. The role of (p)ppGpp in cell biology is to signal stress and to curtail cellular growth functions not needed during stress. In the absence of (p)ppGpp nutritional stress is lethal. Regulation of gene expression by (p)ppGpp occurs at the level of interactions with RNA polymerase. An additional protein, called DksA, is needed as a cofactor. The mechanism of regulation by (p)ppGpp and DksA is unusual in that it does not involve DNA recognition elements other than RNA polymerase itself. Nevertheless, effects of (p)ppGpp and DksA and RNA polymerase are promoter-specific. [unreadable] The DksA protein has two known structural mimics: GreA and GreB. Each of the GreA or GreB proteins can also bind to RNA polymerase without involvement of (p)ppGpp but work at step later than initiation of transcription. GreA and GreB free RNA polymerase that has become arrested during the RNA elongation. Our past work has revealed that GreA also has a new function at the level of initiation. Our work this year has used genetic approaches to define a regulatory network involving (p)ppGpp, DksA and GreA. We can identify a condition (suppression of a dnaK mutant) where a biological activity for the three structural homologs is similar and does not depend on the ability to free arrested RNA polymerase. We also find regulatory activities that are not shared by the three similar proteins and these suggest either additional components or multiple mechanisms. Examples are growth sensitivities that differ for the three proteins depending on the presence or absence of (p)ppGpp. Bacterial motility is another example. It requires GreA and ppGpp but not DksA. Elevating DksA reverses the defect due to the absence of ppGpp but not the one due to GreA. GreB has no effects. We are now using genetic screens with multicopy as well as insertion libraries to find new genes that mimic or perturb regulatory phenotypes of GreA, DksA and (p)ppGpp.