Tet(O) cryoEM structure is out!

So it is finally out: the cryoEM structure of Tet(O) on the ribosome we have collaborated on with Joachim Frank's lab is finally published on in Nature Communications. Tet(O) is a bacterial translational GTPase that clears the ribosome from tetracycline antibiotic, and structural data provided in the paper shed light on the mechanism of Tet(O)-mediated resistance. Recently cryoEM of Tet(O)'s close relative, Tet(M) was published by Beckmann and Wilson labs, so now one can compare the two. And yes, they look very similar. No surprise there.

References:

Li et al. Nature Communications (2013) PIMD: 23403578

Dönhöfer at al. PNAS (2012) PIMD:  23027944

GDP and SRL don't mix

Translational GTPases run the ribosomal cycle, and the ribosome talks back - it recruits the GTPases when it is a certain state, affects trGTPase's affinity to G nucleotides and activates the GTP hydrolysis when needed. Using Isothermal Titration Calorimetry we showed that binding of GDP nucleotide and of SRL rRNA element to translational GTPases IF2 and EF-G are mutually exclusive. This suggests a neat mechanism for the destabilisation of the ribosome-bound GDP form of the GTPase: the moor has done his duty, the moor can go.

Due to the technical limitations, the ITC experiments were performed with a 27-nucleotide long RNA piece mimicking the rRNA element as a model. In order to place our results in the framework of the ribosomal cycle we need experiments with the whole ribosome. 

References:

Mitkevich et al., Scientific Reports 2012 2:843, PIMD: 32150791

First PhD defence in the lab

Our first PhD defence took place on November 2d, 2012. Viktoriya Shyp has defended her work "G nucleotide regulation of translational GTPases and the stringent response factor RelA". Mike Cashel, the discoverer of ppGpp, served as opponent.

Hurray to Vika!

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

Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

Most of the mitochondrial proteins are imported from the cytoplasm, with only a small fraction (about 1%) encoded in the mitochondrial genome. Import is mediated by two complexes: TOM (transporter outer membrane) and TIM (transporter inner membrane). We have a pretty good idea about the players involved in mitochondrial protein import, but we have very little idea about the dynamics of TOM/TIM movement in the mitochondrial membrane.

We tried addressing this question using single molecule fluorescent microscopy in isolated yeast mitochondria. What we see is that Tom40, the central component of TOM complex, is highly confined (i.e. restricted in terms of aerea it can sample) but within its confinement it moves pretty rapidly.

References:

Kuzmenko et al., Scientific Reports (2011) 1:95

The RelA/SpoT Homolog (RSH) Superfamily: Distribution and Functional Evolution of ppGpp Synthetases and Hydrolases across the Tree of Life

Stringent response is run by the RSH (RelA / SpoT Homologue) proteins, but there are more RSHs then just these two. Usually researchers were finding them using an ad hoc approach: take your favorite bug you worked with for 10 years, blast its genome with RelA gene, find anything that looks like RelA, test  it.

Finally there is a proper analysis of RSHs across the tree of life: The RelA/SpoT Homolog superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life by Atkinson GC, Tenson T, Hauryliuk V, PLoS ONE, 6(8): e23479. 

Here is the take home message:

• there are loads of different RSHs out there: we identified 30 subgroups! 
• all the RSHs out there are now are classified (for now, that is. New genomes are coming out every day, damn the progress!).
• Archaea, Bacteria, Eucaryotes: they all have RSHs. I repeat: Archaea too.
• there are the long RSHs (i.e. Rel, RelA and SpoT) and there are the short ones. 
• The short ones have either ppGpp synthesis or ppGpp hydrolysis domain. The long ones have both, but not always both are functional.
• by comparing the long ones vs the short ones we identified residues potentially involved in the inter-domain cross-talk in the long ones (the short ones have only one domain thus there is no cross talk there!).

The bottom line: if you work on a strange RSH protein from a strange bug, do check out our paper. 

Fig. 1.  Maximum likelihood phylogeny of the ppGpp hydrolase domain.  Subgroups are labeled and shading behind the branches shows the most common domain structure observed for those groups, as per the legend in the inset box. Symbols on branches indicate bootstrap support, as per the inset box.

Hurray to Gem! 

Single-molecule investigations of the stringent response machinery in living bacterial cells

Wikipedia: "reductionism, an approach to understanding the nature of complex things by reducing them to the interactions of their parts, or to simpler or more fundamental things". This approach was very successful in unrevealing the basic mechanisms of biological systems. Modern biochemistry is reductionism in its pure form: we purify individual components, mix them together in a test tube and make this in vitro system jump through the hoops and this way we learn how it works. Then we extrapolate what we learned from the in vitro system to the cell, and test our model in vivo: overexpress some components, knock-out the other, introduce mutations etc.

However, sometimes producing in vitro system is not feasible, either because it is to laborious or because we simply do not know what are the components. A good solution would be then to do biochemistry, but... inside the living cell. This approach became technically feasible in the recent decades, and was highly successful in cracking these hard problems for which in vitro investigations are just not cutting it. In vivo biochemistry relies on labeling the protein (proteins) of interest with a fluorescent tag, usually a GFP derivative, and then following its movement inside the living cell on the single molecule level. Movement of the protein can tell us about its functional cycle: binding to a partner will slow its diffusion, for instance.

Now this approach was applied to investigation of the stringent response (I have discussed this fascinating bacterial adaptation system quite at length here). In short, when bacteria are starving for amino acids, they accumulate deacylated tRNAs. These bind to the ribosomal A-site, and this situation is sensed by a protein called RelA, which starts producing alarmone molecule ppGpp. One important thing about RelA functional cycle is that it has two states with distinctly different difusion properties: ribosome bound and free.

This was taken advantage of in the recent paper by English at al. RelA was labelled with a fluorescent GFP variant and its diffusion was followed at ms time resolution. Indeed, inactive RelA turned out to be tightly associated with the ribosomes and diffusing slowly (Fig. 1). However, when stress was induced, either by amino acid limitation or by the heat shock, RelA fell off the ribosome and started moving about much, much faster (Fig. 1).

It is known that under these conditions RelA is enzymatically active and produces ppGpp. Since active RelA seems to spend its time off, rather than on the ribosome, it was suggested that ppGpp production is happening off the ribosome as well. And this is a rather unique mechanism for a ribosome-associated factor. Usually on the ribosome is when the protein is active: RelE binds to the ribosome and cuts the mRNA, EF-G binds, hydrolyses GTP and translocates A and P site tRNAs, ricin binds and cuts the ribosomal RNA.

Fig. 1. MSD (Mean Square Displacement) analysis of the RelA diffusion in vivo. Diffusive behavior of active and inactive RelA is compared to that of ribosomes carrying fluorescent label on L25 protein (green triangles) and freely diffusing protein mEos2. Insert shows the difference in the individual trajectories of active (right trajectory) and inactive (left trajectory) RelA.

Now, of course, this mechanism of RelA has to be tested by other methods. As any approach, single molecule tracking in its current form has its limitations, and the biggest one is the labels used, GFP in this case. RelA fused with GFP is not RelA, it can behave somewhat different.

PS: and now this story was covered in the news! HFSP and UppsalaBio (in Swedish). Also it is covered as a Research highlight in Biopolymers.

PPS: a great review of the single molecule investigations in vivo just came out in Nature: Gene-Wei Li and Sunney Xie (2011). Central dogma at the single-molecule level in living cells. Nature, 475, 308-315 PIMD 21776976. Too bad, we are not mentioned!

PPPS: this blog post is covered in The MolBio Carnival #13!

PPPPS: and now our paper made it to F1000.

References:

Xie XS, Choi PJ, Li GW, Lee NK, & Lia G (2008). Single-molecule approach to molecular biology in living bacterial cells. Annual review of biophysics, 37, 417-44 PMID: 18573089

Potrykus K, & Cashel M (2008). (p)ppGpp: still magical? Annual review of microbiology, 62, 35-51 PMID: 18454629

Gallant J, Palmer L, & Pao CC (1977). Anomalous synthesis of ppGpp in growing cells. Cell, 11 (1), 181-5 PMID: 326415

Brian P. English, Vasili Hauryliuk, Arash Sanamrad, Stoyan Tankov, Nynke H. Dekker, and Johan Elf (2011). Single-molecule investigations of the stringent response machinery in living bacterial cells PNAS 108(31), E359-364 PIMD: 21730169 and the PNAS Author Summary

Mendeley group on stringent response

+1: An ancient family of SelB elongation factor-like proteins with a broad but disjunct distribution across archaea

Yay, one more paper is accepted: An ancient family of SelB elongation factor-like proteins with a broad but disjunct distribution across archaea. Gemma C Atkinson, Vasili Hauryliuk and Tanel Tenson, BMC Evolutionary Biology 2011, 11:22.

The nitty-gritty: we found a SelB relative which is not likely to bind and deliver selenocysteinyl-tRNA. It does something else, and we do not know what. tRNA binding domain and ribosome-binding interfaces are intact, but the G domain is messed up, so it might be a translational GTPase with no GTPase activity! For more details check out Gem's blog or... just read the paper, it is open access!