Differential regulation by ppGpp versus pppGpp in Escherichia coli

Area of Science:

• Molecular Biology
• Microbiology
• Biochemistry

Background:

• Guano sine pentaphosphate (ppGpp) and guanosine tetraphosphate (pppGpp) are critical second messengers involved in bacterial responses to nutritional stress.
• The distinct regulatory roles of ppGpp and pppGpp have remained largely unexplored due to the lack of methods to experimentally control their relative intracellular concentrations.

Purpose of the Study:

• To investigate the differential regulatory effects of ppGpp and pppGpp in bacterial cells.
• To elucidate the distinct biological functions of these two related signaling molecules under stress conditions.

Main Methods:

• Engineered Escherichia coli strains to preferentially accumulate either ppGpp or pppGpp using Streptococcal (p)ppGpp synthetase fragments.
• Manipulated the expression of E. coli GppA, a hydrolase that converts pppGpp to ppGpp, to fine-tune nucleotide levels.
• Conducted in vivo and in vitro experiments, including structural determination of E. coli RNA polymerase-σ(70) holoenzyme complexes with ppGpp and pppGpp.

Main Results:

• Demonstrated that pppGpp is less potent than ppGpp in regulating key cellular processes, including growth rate, RNA/DNA ratios, rRNA P1 promoter transcription, threonine operon activation, and RpoS induction.
• Determined the crystal structures revealing that both ppGpp and pppGpp bind to a common site at the interface between the β' and ω subunits of the E. coli RNA polymerase-σ(70) holoenzyme.

Conclusions:

• Established that ppGpp and pppGpp exhibit distinct potencies in modulating bacterial stress responses, with ppGpp being the more potent regulator.
• Provided structural insights into the binding of these nucleotides to RNA polymerase, suggesting a common mechanism of action at the enzyme's interface.