ppGpp regulates numerous targets, and now we added one more: the stringent response factor RelA itself. Using an in vitro stringent response system we showed that ppGpp dramatically increases the turnover rate of RelA, both is the system where RelA is activated by the ribosomes (both naked and programmed with tRNA and mRNA) and in the system where RelA is activated by the ribosomal protein L11 alone.
Figure 1: RelA activation in the 70S-driven in vitro system upon addition of ppGpp
We did 70S and L11 tittrations and demonstrated that ppGpp increases RelA's kcat, making it a more efficient enzyme:
Figure 2. RelA activity as a function of the 70S concentration in presence and absence of ppGpp
What next? First off, we do not know where ppGpp binds and how it regulates RelA on the mechanistic level. Second, since there are at least 30 groups of the RSH proteins, we will figure out which are activated by this mechanism, and which are not. This will provide us some vital clues for understanding the computational properties of the stringent response system. Third, after this in vitro result it is instrumental to show the ppGpp-mediated activation in vivo.
PS: and now our paper got covered as a Research Highlight in Nature Chemical Biology. Yay!
References:
Shyp et al., EMBO Reports (2012) doi: 10.1038/embor.2012.106.
PIMD: 22814757
We did 70S and L11 tittrations and demonstrated that ppGpp increases RelA's kcat, making it a more efficient enzyme:
Figure 2. RelA activity as a function of the 70S concentration in presence and absence of ppGpp
What next? First off, we do not know where ppGpp binds and how it regulates RelA on the mechanistic level. Second, since there are at least 30 groups of the RSH proteins, we will figure out which are activated by this mechanism, and which are not. This will provide us some vital clues for understanding the computational properties of the stringent response system. Third, after this in vitro result it is instrumental to show the ppGpp-mediated activation in vivo.
PS: and now our paper got covered as a Research Highlight in Nature Chemical Biology. Yay!
References:
Shyp et al., EMBO Reports (2012) doi: 10.1038/embor.2012.106.
PIMD: 22814757
I've read your paper, and I am curious about the effect of pppGpp? Does it similarly stimulate synthesis? It appears that you have only tested tetra phosphate in your paper.
ReplyDeleteHi Jessica,
ReplyDeletewe tried purifying pppGpp for in vitro work, but it seems rather unstable, so we dropped this direction. I would guess it will activate, but weaker - just as pppGpp has weaker effects on transcriptional regulation.
Cheers!