Integration Of Macromolecular Synthesis

Child Health And Human Development

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Abstract

We study the coordination of global patterns of bacterial gene expression by nutrient availability with focus on the roles of 3'-pyrophosphorylated analogs of GDP and GTP, collectively abbreviated as (p)ppGpp. Limiting growth for any of a variety of nutrients leads to changes (p)ppGpp levels that provoke regulatory adjustments at levels of transcription, translation, and metabolism. This year we explored a recent discovery that regulation of transcription by (p)ppGpp requires the DksA protein. The DksA protein functions as a co-factor for (p)ppGpp - RNA polymerase (RNAP) interactions. The coiled-coil finger of DksA is believed to penetrate the secondary channel of RNAP then to amplify regulatory effects by stabilizing (p)ppGpp binding near the catalytic center. We asked if other secondary channel binding proteins that share structural features with DksA can alter RNAP regulation through mutually competitive interactions with DksA.Two such structural homologs are the transcription elongation factors, GreA and GreB. Both are known to relieve arrested transcription by cleaving nascent RNA chains that have backtracked out of reach of the catalytic center. Using a (p)ppGpp regulated ribosomal RNA promoter, indeed we found in vivo regulation was sensitive to balanced ratios of DksA: GreA and to a lesser extent DksA:GreB. With a purified transcription system we learned that GreA provoked a conformational change of RNAP DNA open complexes that was abolished at a later step in initiation by DksA.We conclude that studies of a specific promoter do reveal mutual interactions between secondary channel interacting proteins.[unreadable] The generality of this finding is being tested as follows. We found long ago that E. coli deleted for its capacity to form (p)ppGpp gains nutritional requirements for several amino acids (ILVTFHS). These requirements can be abolished by suppressor mutations in RNAP rpoB, rpoC and rpoD subunit genes; these suppressing RNAP alleles are understood as altering RNAP in a manner mimicking the effects of the presence of (p)ppGpp. Now we have found that reversal of the ILVTFHA requirements can also be achieved by overproducing GreA (and to a lesser extent GreB). Importantly, this activity of GreA (or GreB) only occurs when DksA is deleted. This constellation of observations apparently again reveals the importance of balanced levels of GreA (GreB) and DksA, but on different promoters. Mutant evidence suggest these interactions can be distinguished from the known functions of GreA/B to relieve transcription arrest. GreA mutant residues in the tip of the coiled-coil finger that are unable to relieve arrested transcription have more, not less, potent effects than the native GreA as suppressors of the amino acid requirements of (p)ppGpp-deficient E. coli. We now wish to understand this new function of GreA/B on transcription of promoters involved in amino acid biosynthesis.