catastrophic drop in ribosomal research in 1910

Just found this: http://ngrams.googlelabs.com/

This seems to be a great way to waste a lot of time.

Look at curious spike of interest to tRNA and ribosomes around 1900... this surely means something important. Let us zoom in:

Trends for tRNA and ribosome coincide, so it can not be just a glitch, there should have been some serious research done on translation around the turn on the century... But why did they stop? May be they failed to discover some important components and thus research dwindled?

mRNA? Nah, seems like mRNA was very much a big thing then! How about RelA? Yes! This is it. Now we know - failure to advance translational research around 1900 was caused by failure to discover RelA. Case closed.

specific inhibition of nonsense-mediated mRNA decay by small molecule

The last step of protein synthesis is called translation termination. During this step the stop codon is recognized by the protein factor called "release factor" and finished protein is cleaved off the tRNA. Mutations which cause premature termination (nonsense mutations) lead to shortened protein which is usually defective, and in order to avoid accumulation of these proteins such mRNA are recognized by nonsense mediated mRNA decay (NMD) machinery and degraded.

This is usually a good thing, but imagine that the gene which has this nonsense mutation is very important for survival of the cell, and imagine that the truncated version is still somewhat functional. In this case it would be beneficial to produce it even if it is somewhat compromised: something is better than nothing.

Therefore in some cases NMD is actually a cause of a desease, such as Duchenne muscular dystrophy and cystic fibrosis - over-zealous NMD removes all the damaged mRNAs and no protein product is produced. In this case suppression of NMD is needed.

Treatment with antibiotic gentamicin wich causes error-prone translation is a solution, but unfortunately gentamicin is not specific for NMD and causes errors on all the steps of translation. Specific inhibition of NMD was sought after for many years, and finally a smal molecule which inhibits it was discovered.

The molecule is called PTC124, and we know that it does the trick (inhibits NMD) and works well against the DMD. It was discovered in 2007, and by now we still don't know to what it binds (ribosome? NMD factors?) and what is the mechanism. The reason for that is that it is extremely complicated to construct an in vitro system for studying PTC124 (you will need purified translational and NMD components, and that is a lot of components). So for now we have no idea how it works, but it does!

Update: ...or may be it doesn't!

References:

Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P, Paushkin S, Patel M, Trotta CR, Hwang S, Wilde RG, Karp G, Takasugi J, Chen G, Jones S, Ren H, Moon YC, Corson D, Turpoff AA, Campbell JA, Conn MM, Khan A, Almstead NG, Hedrick J, Mollin A, Risher N, Weetall M, Yeh S, Branstrom AA, Colacino JM, Babiak J, Ju WD, Hirawat S, Northcutt VJ, Miller LL, Spatrick P, He F, Kawana M, Feng H, Jacobson A, Peltz SW, & Sweeney HL (2007). PTC124 targets genetic disorders caused by nonsense mutations. Nature, 447 (7140), 87-91 PMID: 17450125

Manuvakhova M, Keeling K, & Bedwell DM (2000). Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system. RNA (New York, N.Y.), 6 (7), 1044-55 PMID: 10917599

Double life of bacterial elongation factor EF-Tu

Protein biosyntheses is performed by the ribosome and is assisted by a large number of accessory molecules, with several of them belonging to the GTPase class. EF-Tu is a GTPase which is responsible for bringing the amynoacyl-tRNA to the ribosome, and it is one of the most abundant proteins in bacteria.

So abundant, in fact, that it is impossible to explain this abundance by EF-Tu's role in translation. And indeed, recent microscopy investigations has shown how EF-Tu works as a part of bacterial cytosceleton.

EF-Tu works together with another bacterial structural protein, actin-like protein MreB, which froms spirals underneath bacterial membrane. In this tandem EF-Tu leads the formation of more dynamic MreB filaments.

In eucaryotes eEF1A does the job of EF-Tu and delivers the tRNA. It is involved in interactions with the cytosceleton just as it its bacterial counterpart, but instead of MreB it interacts with the filamentous actin, causing it to form bundles.

Bunai F, Ando K, Ueno H, & Numata O (2006). Tetrahymena eukaryotic translation elongation factor 1A (eEF1A) bundles filamentous actin through dimer formation. Journal of biochemistry, 140 (3), 393-9 PMID: 16877446

Defeu Soufo HJ, Reimold C, Linne U, Knust T, Gescher J, & Graumann PL (2010). Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein. Proceedings of the National Academy of Sciences of the United States of America, 107 (7), 3163-8 PMID: 20133608

Vats P, & Rothfield L (2007). Duplication and segregation of the actin (MreB) cytoskeleton during the prokaryotic cell cycle. Proceedings of the National Academy of Sciences of the United States of America, 104 (45), 17795-800 PMID: 17978175

paper of the week: Kuntz et al., PNAS '99

Todays paper of the week is from the late summer of 1999. Better still, is from the University of California... Late 90s, late summer, California - this all sounds marvelous.

I really like papers which start with a question you never thought existed, but it turnes out that it is immediately interesting and even you want to know the answer. So the question here is: what is the maximal possible affinity of a ligand?

The approach of Kuntz and colleagues is simple: they search the literature and plot known affinities (ΔG) vs the number of non-hydrogen atoms for all the tightly biding ligands they can get their hands on.

The trend they get is beautiful: below 15 atoms you get 1.5 kCal per atom, and adding more than 15 atoms does not improve the affinity any more. There are some outliers: metal ions and biotin, for instance. So now we know.

Kuntz, I., Chen, K, Sharp, K, & Kollman, P (1999). The maximal affinity of ligands Proceedings of the National Academy of Sciences, 96 (18), 9997-10002 DOI: 10.1073/pnas.96.18.9997

my first non-scientific post

Modern internet-based humans seem to be trying to externalize as much of themselves as possible. Blogging is nothing in comparison to http://www.evernote.com/ - an attempt to keep all your valuable memories outside of your scull. Surely, it is useful. As Dawkins says, "We should be open-minded, but not so open-minded that our brain falls out".

Yeah, todays blogging and externalization done.

Searching for antibacterials under the lamppost

We all know that we need more antibiotics and the ones we have are no good. We all also know that bugs become more and more skilled in avoiding being killed, and this is no good either.

The question here is why don't we have new antibiotics coming. And it turns out to be real hard to make one. Initially we had a discovered a whole bunch of these from natural components. All these were developed by bacteria and fungi to kill other bacteria and fungi, and these chaps had billions of years to develop these components. We just hijacked their research, really.

When all these started to age because of the pathogens rapidly acquiring resistance, big pharma decided to have a go at making new antibacterials. Hey, we are pretty good at fighting cancer, killing some bugs should be a walkover.

Here is a great example how we fail. By "we" I mean GlaxoSmithKline, who are no amateurs. They constructed a library of constructs, which would produce genes of interest at different expression level - and that would be all the genes in the genome they could clone that way!,  and tested all these against all the compounds they had on their shelves. ...WOW!

Many, many monies later: 10 hits, no leads, no drugs. Game over.

But let us imagine that there would be drugs developed. What next? These would be the last line of defense, and will be proscribed very, very rarely. This creates a problem of profit in the antibiotics R and D: even if you develop a new antibacterial, there will be no money coming you way.

Well then, pharma can not help us... How about academia?

There is some hope there. A couple of new approaches has emerged.  One is grafting one antibiotic scaffold on another, creating a hybrid werwolf antibiotic, which kills like hell. However, majority of the academia peoples just figured out that anything is an antibiotic target, and that anything is an antibiotic, and they started killing their favorite proteins or RNA in vitro. And yes, if you inhibit something important, your bug will die.

RelA is no exception. Several ppGpp analogues were developed as lovely antibacterials: exhibit 1 and  2. (A quick reminder - RelA makes ppGpp from one ATP and one GDP molecule, and ppGpp in turn acts as a molecular messenger remodeling the whole bacterial physiology, thus RelA is very important for bacterial virulence - a great target for an antibacterial indeed!).

Does it look promising? Not at all. It is not enough to inhibit something important. You also need to be specific and you also need to get into the cell. The latter is a very, very big problem - you need to follow the Lipinski's rule of five, and ppGpp is a very, very bad scaffold to start with in this case: loads of hydrogen bond donors, loads of acceptors, too big and not hydrophobic enough.  All bad. Well, the only things that are good is grants acquired and papers published.

So here we are. Big pharma bails out, academia has no idea as to what they are doing. Any chances of our survival?

Yes. Small start-up pharma packed with academia-derived know-how. Rib-X, Tetraphase. Pray for them, please.

References:

Payne DJ, Gwynn MN, Holmes DJ, & Pompliano DL (2007). Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nature reviews. Drug discovery, 6 (1), 29-40 PMID: 17159923

Charest MG, Lerner CD, Brubaker JD, Siegel DR, & Myers AG (2005). A convergent enantioselective route to structurally diverse 6-deoxytetracycline antibiotics. Science (New York, N.Y.), 308 (5720), 395-8 PMID: 15831754

Mendeley group on stringent response

Biochemistry and Evolution: heterologous systems

We have loads of different critters co-inhabiting with us, and this can be very useful if you need to publish some papers and progress your career. There are at least 2 ways of doing so.

First, you can do phylogenetic analysis of these and ask questions, such as what is conserved in particular protein? Can we relate these conserved residues to some conserved function? What is not conserved? How is this protein related to other proteins? Can we retrace functional evolution via sequence evolution and vice versa, can we predict some functions for the not yet well studied protein knowing something about its sequence? Gem here is talking about stuff like that.

However, there is a problem with this sort of approach. You end up presenting your data in a format that can be criticized by reviewers or readers, or both. They might wonder about your bootstrap values, accuse you of LBA and in general scoff your alignments and so on. Also, you endup testing your hypotheses, or having them tested by other people, and there is a possibility of being wrong when you test things you predict.

Therefore there is an alternative way of benefiting from the diversity of live in order to produce papers. That would be using heterologous systems. This is how it goes.

You take an interacting protein pair from 2 bugs, A and B. It does not matter what bugs are these. Some bugs. As an example you can take proteins EF-G and RRF which split the ribosome after it has finished making the protein. Then you mix them and see if they can do the job in different combinations (AA, AB, BA and BB). If they all work, then you take bug C, and repeat it until you find a combination that does not work. And if you are not bored, you can take bugs D and F and do it again. So far you produced two papers.

The only question remaining is - did you learn anything? From the evolutionary point of view your result is boring - you have found some sequence co-variation, which results in factors from the same organism working together, but not cooperating with a stranger. This is an obvious result which so far generated no hypotheses (having an alignment on the side could generate one though - looking for the co-variation in the 2 proteins would suggest where the differences are).

And when we talk about alignments, we are talking about multiple ones. Aligning 2 genes from the bugs A and B you study has very little information about the conservation of the differences you are looking at (but this is exactly what is done in the EF-G / RRF paper cited above and in the later paper from the same group working now on IF2 from E. coli and bovine mitochondria - heterologous system strikes again!).

References:

Having a tree to go with alignment will make it all even more informative, cause then you can see the directions of sequence changes, but hey - now we are getting in trouble! We are about to do it properly.

Rao AR, & Varshney U (2001). Specific interaction between the ribosome recycling factor and the elongation factor G from Mycobacterium tuberculosis mediates peptidyl-tRNA release and ribosome recycling in Escherichia coli. The EMBO journal, 20 (11), 2977-86 PMID: 11387230

Seshadri A, Singh NS, & Varshney U (2010). Recycling of the posttermination complexes of Mycobacterium smegmatis and Escherichia coli ribosomes using heterologous factors. Journal of molecular biology, 401 (5), 854-65 PMID: 20561528

GFP-based quantification of proteins in the cell - don't try that at home!

This is something that come out of our tracking experiments in J. Elf lab. I guess we will never publish any quantitive account of it, so it guess it constitutes perfect blogging material.

GFP and its derivatives are widely used to label and track proteins in live cells. This way people follow localization of the protein of interest and accessing changes its concentration.

And here is where the fun begins. GFP has a fluorofor that is bringt only in a) oxidized b) deprotonized state. Therefore redox potential and pH of the cellular environment have profound effect on the brightness of GFP. Cultural media per se can change GFP behavior dramatically, and this is something outside of the cell...

Another problem with GFP is that it can radically affect stability of the fusion protein. We are not even talking about function here, we are talking about the number of molecules per se.

While struggling with our Dendra2 GFP variant and RelA_Dendra2 fusion in vivo we observed all these problems constantly. Unhappy cells were dark, with GFP in the dark state (pH? redox? you simply don't know!) and the numbers of proteins you detect were invariably much lower than what is estimated for the wt, un-tagged RelA.

But all this still does not stop brave souls from using GFP for single molecule quantification of bacterial proteins en masse. Whole library of YFP (yellow fluorescent protein) fusions was created and their copy number as well as diffusion characteristics were analyzed, and then systems biology happened to the dataset. No one knows what exactly does it all mean and how the numbers of detected YFP-fused proteins relate to numbers of the wt proteins, but this is details.

References:

Bogdanov AM, Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov S, & Lukyanov KA (2009). Cell culture medium affects GFP photostability: a solution. Nature methods, 6 (12), 859-60 PMID: 19935837

Taniguchi Y, Choi PJ, Li GW, Chen H, Babu M, Hearn J, Emili A, & Xie XS (2010). Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science (New York, N.Y.), 329 (5991), 533-8 PMID: 20671182

Brian P. English, Arash Sanamrad, Stoyan Tankov, Vasili Hauryliuk, & Johan Elf (2010). Tracking of individual freely diffusing fluorescent protein molecules in
the bacterial cytoplasm arXiv q-bio arXiv: 1003.2110v1

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

Bacteria grow in order to grow, this is what they do. And they try to do so as efficiently and fast as possible. This results in two things.

First, they mostly produce stuff for producing more stuff, and that would be ribosomes for making proteins. Ribosomes and other translational components (tRNAs, protein factors) constitute the bulk of  bacterial dry mass.

Tightly connected to this ribosomal expansion phenomenon is the fact that bacteria really, really need to regulate production of their so-called stable RNA (ribosomal RNA (rRNA) and tRNA (shame on me... transfer? transport? well, you get the idea!). Regulation of the stable RNAs is regulation of bacterial growth, it is that simple (really not! but let us simplify). And in order to regulate production of these RNA they have stringent response and specifically RelA protein.

In reality they have stringent response in order to cause me pain. The good thing is that they do not cause pain exclusively to me, although most of the other people who were working on stringent response have by now either moved to other fields, or nearing retirement and thus are nor so afraid of working on the stringent response any more, or died. I can not switch fields now, nor can I retire yet. Alternatives?

For all the major steps and players of translational machinery we are now enjoying massive amounts of high quality data. Structures are available, mechanisms are described as very, very detailed kinetic schemes etc. For RelA this is not the case.

We have a very, very vague idea about the mechanism and we have an x-ray structure of the truncated protein. However, we have no cryo-structure of RelA bound to the ribosome, as we have do for the majority of other translational factors (and usually in many different complexes!), nor do we have any kinetic schemes dissecting the mechanism into individual steps. All we have is bulk enzymatic assays providing Km and kcats, but this is really not in the keeping with the the advancement of our understanding of all other aspects of translation and its regulation.

Why is that so? Next time I feel like blogging, I will try addressing this question.

Mendeley group on stringent response

Papers, titles, exclamation marks

Using exclamation marks in scientific papers is a controversial issue. Some people do use them in titles, some - don't. It is down to the keyboard layout you are using, really. Well, if exclamation marks are kosher, then how about this:


A new paper from Yusupov: "OMG! OMG! Yeast 70S ribosome at 2.5 A resolution, WOW!" the real WOW of the discovery that it is actually 70, but not 80S! Only x-ray could determine that. And only at 2.5 A, OMG!


And this  - "Bacterial ribosomal recycling is accomplished by concerted action of EF-G and RRF, LOL!" This one is from Rodnina.

Or imagine a letter to editor. Say, Science. "Arsenic-based life - WTF!?" Well, WTF indeed.

That was all for today.