Positive feedback control of E. coli RelA by its product ppGpp

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

Proline residue in L11 as a key regulator of translational GTPases?

The ribosome is run by translational GTPases. Translational GTPases, in their turn, are regulated by the ribosome. They all bind in the same region (GAC, GTPase associated center) of the ribosome. In bacteria the GTPase binding site consists of a couple of rRNA elements: SRL (sarcin-ricin loop) and thiostrepton loop and several ribosomal proteins:L7/L12 stalk (L10 and L7/L12) and L11.

The latter is the main hero of a fresh paper in Nature Structural and Molecular Biology by Wang and coworkers. They show that bacterial translational GTPases (such as EF-G) when binding to the ribosome act as peptidyl-prolyl cis-trans isomerases (PPIases) driving isomerisation in the conserved residue in the ribosomal protein L11. This isomerisation, in turn, transmits signal to the ribosomal protein L7 /L12 - something that is necessary for efficient GTPase cycling on the ribosome.

I like L11 - it is a key protein for stringent response, and without it stringent factor RelA does not work, as was shown using E. coli mutants lacking L11 (Dabbs J. Bac 1979). These mutants are perfectly viable, but grow ten times slower then the wild type E. coli, most probably due to defects in the ribosome assembly (Hampl et al. JBC 1981). The very viability of the L11 knock-out strains tells us that L11 is not the key for keeping the ribosome running. In fact, less than a half of ribosomal proteins can be knocked-out in E. coli (22 out of 54, Shoji et al. JMB 2011), making L11 one of the less-important ones... and keeping an eye of the translational GTPases is definitely not one of the less-important functions!

This seems to be bit paradoxical - a ribosomal protein that is dispensable involved in something that is very central for protein biosynthesis. It gets even more fascinating when you look at the evolutionary aspect of the story (Gem Atkinson does that in her blog). Wang and colleagues managed to map  the PPIase site of EF-G.  As they show PPIase activity is universal for all the bacterial translational GTPases they tested, and the PPIase site is, surprisingly, quite a variable region of the G domain! So, do they all reinvent the weel separately? This is all most peculiar.

References:

Wang et al. A conserved proline switch on the ribosome facilitates the recruitment and binding of trGTPases. Nat Struct Mol Biol (2012) PIMD: 22407015