Stringent response - why are we still in the middle ages? Part II.

This is a continuation of my previous post on this subject, see here.

So here are some more reasons for investigation of stringent response being such a painful and slow-going venture. Let us go through the approaches we have.

Microbiology

Here we have a bunch of problems. Stringent response is very central and messing with it has very pleortopic effects which is not helping. Doing knock-outs of RelA and SpoT is a tricky business, and compensatory mutations can and do arise. However, I would say that microbiological data are the best part of the data available, especially since different model organisms were studied recently and work on different RSH proteins is done, linking RSH proteins to different targets (see Battesti 2005, Vinella 2005, Persky 2009,  Lemos 2007, Gatewood 1010, and Maciag 2010).

Definitely, all the best things in the stringent response field are done using the microbiological approach. Just as they were done 10 years ago. Or 20. This is a bit sad.

Structural methods

Here we have more serious problems and less succes stories. RSH proteins are horrible to work with, they tend to precipitate a lot. Therefore the only structural insight we have so far is x-ray of the truncated Rel protein (Hogg 2004) - but this is only one representative of the RSH family which is actually much bigger than what is presented in Mittenhuber 2001! we have no idea as to how RelA or SpoT look like, we can only guess using homology modeling using the Rel structure.

No cryoelecton reconstructions are available, and the reason is simple: in order to have a good cryoEM sample, you need to have high occupancy (high % of RelA-bound ribosomes in this case), and for that to happen RelA needs to bind really tight (working concentrations are roughly 50 nM 70S). Of course, one can hammer the ligand (RelA in our case) in just by increasing its concentration - it is simply a matter of  Le Chatelier's principle! - but again, you add loads of RelA and it precipitates. Bummer!

How about crosslinking? This is a bit more promising, but still - there are issues. RelA:tRNA crosslink is a hard one, since standart procedure of modifying the by oxidation tRNA CCA which was used for crosslinking with FMT (Hountondji 1980, Gite 1997) does not work for RelA: it does not want to interact with with the modified tRNA any more (Sprinzl 1976)!

Biochemistry

As I say, RelA is a nasty protein to work with. Rel seems to be a bit better, and considerable progress was done using it (Sajish 2009, Avarbock 2005). Some good stuff was done with E. coli protein too (Jenvert 2007), but unfortunately this lab has already folded. Actually that seems to be a trend - it seems to be unlikely for the lab to continue working on RSH proteins in vitro for a long time. Very nice proteins, yes.

The main problem here is that all biochemistry is done with greatly simplified in vitro systems - people use just 70S and mRNA, and tRNA; no initiation or pre-transclocation complexes which would be much more of a natural substrate. This is the direction where we are going.

As to SpoT, no one can purify nowadays, the only reports of people doing this (and we do not really know what they purified since no masspec of the SDS PAGE band was done) are dating from late 70s (Ma 1979). And this really hinders this direction.

Single molecule methods

Well, see this. SPT investigations of RelA are coming, but and the main problem here is - surprise! - microbiology. Getting the strains right is really hard and takes lots of effort and control, and it is very, very easy to end up working with some very strange mutant.

What we really need there is some sensor for in vivo concentrations of ppGpp in the individual cells so that we could combine RelA tracking data with ppGpp production. There is a sensor for ppGpp (Rhee 2008), but getting inside E. coli... that would be a tricky one.

Hight throughput approaches: microarrays, libraries...

They exist, and things are done (Traxler 2006, Traxler 2008, Durfee 2008, Traxler 2011 and Balsalobre 2011). This seems to be a very promising approach able to resolve kinetics of stringent response developing during starvation: which geners are affected, in which order. Combined with mutational analysis it really holds promise for in vivo investigations of stringent response (for more detailed discussion see here).

Mendeley group on stringent response