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