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

iGEM2011: GFP-based readout for ppGpp concentration in vivo

Measuring ppGpp concentration in individual living cells with good temporal resolution would be great. I've been musing about a possibility of doing that using RNA aptamers, but that's just musing. It seems like am iGEM team from the University of Trondheim tried setting up a GFP-based reported system, and this system is, maybe, possibly, probably, working. Somewhat. 


Unfortunately there is no publication. However, there is a popular article about the whole affair, in Norwegian and there is a short report on the iGEM webpage. 


Apropos to the technical issues that are listed in the original report, such as dramatic leakage of the GFP reporter in the absence of stress stimuli, there are several conceptual concerns. First, the system is based on translation of the GFP reporter during the stringent response, and during the stringent response translation is strongly inhibited. Second, GFP (they use red version of it, mCherry) has to mature in order to become bringt, and that takes some time - from minutes to hours, depending on the conditions and what sort of GFP variant it is. For mCherry maturation time is 15-40 minutes, and this is comparable with E. coli generation time. Therefore, first, one would expect a very pronounced lag before the SR is engaged and the corresponding readout and, second, all the fluctuations in the ppGpp concentrations happening on the timescale below 10s of minutes will be averaged out. Third, GFP is very stable, so this reporter system will have severe memory effects - once the cell has committed to stringency, it will produce GFP, and even though stringency is reversed, GFP will stay. Maybe it is possible to turn this into a feature, but I am not sure how. And, lastly, brightness of the GFP depends on the pH and redox potential of the environment, and these things change in the stressed cells.

More fun with fun12... right, that was a dreadful joke

I like translational GTPases. I particularly like bacterial ones. Hell, I am titrating one with GTP as we speak, so I am partial. However, eukaryotic translational GTPases are also OK, particularly the ones that have bacterial homologues.

One of these is eukaryotic initiation factor 5B, eIF5B, a homologue of bacterial initiation factor 2, IF2. In S. cerevisiae eIF5B is called Fun12. eIF5B is a GTPase, and translational GTPases are tightly regulated. During translation initiation Fun12 is involved in the subunit joining, and GTP hydrolysis is coupled with factor's release from the initiation complex. 

Now it turnes out that Fin12/eIF5B has another function outside of translation initiation: together with the 60S subunit it proofreads the ribosomal assembly (Lebaron et al. 2012 and Strunk et al. 2012). Assembly of the ribosomal subunits is a multistep process, with r-proteins binding is certain sequence, assisted by various assembly factors, with rRNA being cut and remodeled. One of the steps is cleavage of the 18S rRNA by the Nob1 RNAse. And this stage, apparently, is stimulated by Fun12. Bacterial Fun12 homologue, IF2, is GTP-dependently rearranging the ribosomal structure by inducing intersubunit rotation, so the same process is now suggested to play the role in 40S maturation in yeast via driving the ribosome in Nob1-susceptible state.


In eucaryotes translation-incompetent ribosomes undergo so-called nonfunctional rRNA decay, NRD. By linking functionality on Fun12 binding and activation via consecutive 60S binding, translational functionality is linked to rRNA processing, providing another safety net making sure that only functional ribosomes are involved in translation, and the nonfunctional ones are rapidly cleared out.

References:

Lebaron et al. Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits. Nature Str. Mol. Biol. 2012 in press PIMD: 22751017


Strunk et al. A Translation-Like Cycle Is a Quality Control Checkpoint for Maturing 40S Ribosome Subunits. Cell 2012 vol. 150 (1) pp. 111-121 PIMD: 22770215

One more '-omics' analysis of the stringent response: BIBLIOMICS

The stringent response is confusing, no doubt about that. I personally get exceedingly confused when I read in vivo papers from the 80s. I somehow hope that there should be hidden gems there, and it is just my stupidity that stops me from discovering these.... so I try... and I get confused. In vivo data confuse me, and in vivo data from the 80s.... I am lost.

Importantly, I always try to read one paper at a time, maximum ten, rarely more then twenty. Imagine what happens if one would read them all? And how, I wonder, how would one call this sort of thing? Wonder no more; enter Carneiro and colleagues. What they did, they collected the whole bibliome about the E. coli stringent response and analyzed it in attempt to gain a birds-eye view, providing 'a more systematic understanding of this cellular response'. 

They summarize the nitty-gritty of the stringent response in the magnificent Figure 1.

 The sheer number of mistakes they make is owe-inspiring and truly shows the power of the high-throughput learning. It is OK that tRNAs have their anticodons on one the 3' (or is it 5'?) end and amino acids are attached where the anticodons should be. Fine, that the amino acid moieties are outside of the ribosome when the tRNAs are attached. Fine, that there is no way in hell one can figure out there are the A- , P- and E-sites of their ribosomes, and, surely, it would be nice to be able to know position of at least the A-site when we are talking about the stringent response. But what is truly fabulous, is that the ribosomal protein L11 is part of the small ribosomal subunit (L... the letter L is giving us a hint... large, maybe?...). Since RelA is interacting with L11, so this causes a bit of a trouble, and RelA ends up on the small subunit as well (ah, never mind this paper). In this awkward position RelA has no chances of inspecting the CCA of the A-site tRNA, but maybe it is for the best, given how messed up the tRNA already is and that we have no idea where the A-site could be...


The bird-eye view allows Carneiro and collegues to make some information-packed generalizations. I am not sure what we learn this way, but the figures speak for themselves:

Rrrright, the dashed line overtook the solid one and they never crossed, therefore the ratios between the blue and red bars changed with time... I want some error-bars, then it will be really, really nice and scientific.


But it is not only the figures that are great. The text is awesome as well. 'Later, in 1980, the ppGpp level was found to be controlled by the SpoT enzyme via GTP hydrolysis activity (PMID: 6159345)'. GTP hydrolysis! By SpoT! Yay! 'The 50S ribosomal subunit protein L11 has been indirectly implicated in the feedback inhibition of (p)ppGpp, because ribosomes lacking this protein are unable to stimulate the synthesis of these nucleotides (PMID:11673421; PMID:17095013) [39,61].' Feedback inhibition! This redefines the meaning of feedback... Wow! No wonder L11 migrated on the large subunit... 


Being subjected to such a monumental degree of confusion, the authors started expressing themselves in a most peculiar way; for the lack of a better word I would call it 'cautious': 'As a result, it was possible to perceive the relevance of specific translation GTPases known to be inhibited by (p)ppGpp nucleotides'. 'Studies showed that (p)ppGpp inhibits translation by repressing the expression of ribosomal proteins and also potentially inhibiting the activity of the particular proteins'. 


I certainly hope that soon the authors should take on the mighty ribosome. There are many more papers in the ribosomal bibliome, and the level of confusion might be even higher. I would also recommend some 3D plots!

 References:

Carneiro, S., Lourenço, A., Ferreira, E. C., & Rocha, I. (2011). Stringent response of Escherichia coli: revisiting the bibliome using literature mining. Microbial informatics and experimentation, 1(1), 14. doi:10.1186/2042-5783-1-14 PIMD: 22587779

One more crazy little thing

Stringent response is run by a family of proteins called RelA-SpoT Homologues, RSH, and these come in two flavors: the long ones and the short ones. The long ones have both ppGpp synthesizing and ppGpp hydrolyzing domains, and either both are active, with synthesizing being the dominant one (that would be the ancestral Rel) or both are active, with hydrolyzing being the dominant one (SpoT) or only the synthesizing one is active (RelA). The short RSHs are more variable. They have only one of the domains, so they can either synthesize (SAS, small alarmone synthetase) or hydrolyze (SAH, small alarmone synthetase) ppGpp. What is fun, is that in addition they can have other domains, sometimes with very peculiar functions.

A very peculiar SAS was characterized recently by Maya Murdeshwar and Dipankar Chatterji. They call it MS_RHII-RSD, or, using terminology proposed in our paper with Gemma Atkinson, actRelMsm. This SAS from Mycobacterium smegmatis in addition to the ppGpp synthetic activity has another one, quite unexpected. It has a dedicated domain capable of hydrolyzing DNA:RNA duplexes via its RNAse H domain.

Quite bizarre, quite.


References:


MS_RHII-RSD:  a dual function RNase HII - (p)ppGpp synthetase from Mycobacterium smegmatis. M. Murdeshwar and D. Chatterji. J. Bacteriology, 2012, in press PIMD: 22636779