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Insights into the mechanism of DksA action at ribosomal RNA promoters in Escherichia coli
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EAN 2940031819395
REGISTERED: 09/19/18
UPDATED: 12/14/18
Insights into the mechanism of DksA action at ribosomal RNA promoters in Escherichia coli.
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In the absence of the dksA gene, Escherichia coli ribosome synthesis is uncoupled from the cellular demand for translation. DksA affects ribosomal RNA (rRNA) transcription initiation, the rate-limiting step in ribosome synthesis, by modifying the RNA polymerase (RNAP)-promoter complex to make it susceptible to regulation by small molecules that change in response to the translational demands of the cell: guanosine tetraphosphate and NTPs. We investigated the mechanism by which DksA changes the


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  • EAN bar code 2940031819395 ξ1 registered March 16 2016
  • Product category is Insights-into-the-mechanism-of-DksA-action-at-ribosomal-RNA-promoters-in-Escherichia-coli Steven-Thomas-Rutherford Electronic Cell

In the absence of the dksA gene, Escherichia coli ribosome synthesis is uncoupled from the cellular demand for translation. DksA affects ribosomal RNA (rRNA) transcription initiation, the rate-limiting step in ribosome synthesis, by modifying the RNA polymerase (RNAP)-promoter complex to make it susceptible to regulation by small molecules that change in response to the translational demands of the cell: guanosine tetraphosphate and NTPs. We investigated the mechanism by which DksA changes the RNAP-promoter complex using genetic and biochemical approaches.;First, we compared activities of DksA and GreB (both have coiled coil domains and bind in the RNAP secondary channel) at rRNA promoters. We found that when overexpressed GreB functionally substitutes for DksA, but the in vivo GreB concentration is too low to affect rRNA regulation in normal cells. DksA possesses other activities that GreB lacks, indicating that, in addition to sharing contacts, DksA has unique interactions with RNAP. Next, DNase1 footprinting was used to identify a specific step in transcription initiation affected by DksA (RPI formation), and substitutions in the RNAP trigger loop, bridge helix and switch regions that altered the response of RNAP to DksA were identified. Finally, we confirmed that the RNAP secondary channel as the DksA binding site using RNA crosslinking, protein-protein footprinting, and by examining properties of RNAPs with substitutions in secondary channel residues.;We propose the following working model for the mechanism of DksA action. DksA binds in the RNAP secondary channel and interacts with RNAP residues that transduce a signal to parts of RNAP that interact with DNA during formation of RPI. This signal is transmitted over 25A across RNAP possibly via the bridge helix and switch regions. As a result, structural changes and/or RNAP-promoter interactions required for formation of RPI are prevented, thus shifting occupancy in the dissociation direction. RPI is highly


References
    ^ Insights into the mechanism of DksA action at ribosomal RNA promoters in Escherichia coli. (revised Dec 2018)

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