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I am a biochemist based in Tartu, Estonia, working on translation and stringent response in bacteria. For more info visit my lab webpage http://www.tuit.ut.ee/hauryliuk/hauryliuklab/Welcome.html
stringent response
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- January 6, 2011
- 09:57 AM
- 471 views
How to swap a gearbox for a new model right on the highway
by Vasili Hauryliuk in stringent response
Protein biosyntheses is central a hub for cellular physiology: proteins are essencial for all the cellular processes. Therefore changing something really important in translational machinery is really hard: you still need producing proteins! Swapping an important translational factor for another one? That sounds impossible, but this is exactly what happend with eEF1A - eukaryotic factor that brings aminoacylated tRNA to the ribosome. Moreover, it happened several times!It was indeed swapped for a similar, yet different protein EFL (EF-Like) several times during eukaryotic evolution. The main difference between EFL and eEF1A is in theis GTPase cycle. eEF1A, just like its bacterial counterpart EF-Tu, needs a specialized factor in order to regenerate it from the GDP to GTP-bound state (GTP Exchange factor, GEF). EFL does not need a GEF, so it is in a sense simpler. Loosing a GEF seems to be a common theme in the evolution of translational GTPases. Mitochondrial EF-Tu lost its GEF (EF-Ts) in Saccharomyces cerevisiae, though retained that in human and S. pombe! Moreover, it is possible to select mutants in yeast eEF1A which would confere GEF-independence, turning into something like EFL.Sometimes regulating GTPases is just too much to ask for and Nature cuts corners.References:Keeling PJ, & Inagaki Y (2004). A class of eukaryotic GTPase with a punctate distribution suggesting multiple functional replacements of translation elongation factor 1alpha. Proceedings of the National Academy of Sciences of the United States of America, 101 (43), 15380-5 PMID: 15492217Rosenthal LP, & Bodley JW (1987). Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu. The Journal of biological chemistry, 262 (23), 10955-9 PMID: 3301847Chiron S, Suleau A, & Bonnefoy N (2005). Mitochondrial translation: elongation factor tu is essential in fission yeast and depends on an exchange factor conserved in humans but not in budding yeast. Genetics, 169 (4), 1891-901 PMID: 15695360Ozturk SB, & Kinzy TG (2008). Guanine nucleotide exchange factor independence of the G-protein eEF1A through novel mutant forms and biochemical properties. The Journal of biological chemistry, 283 (34), 23244-53 PMID: 18562321... Read more »
Keeling PJ, & Inagaki Y. (2004) A class of eukaryotic GTPase with a punctate distribution suggesting multiple functional replacements of translation elongation factor 1alpha. Proceedings of the National Academy of Sciences of the United States of America, 101(43), 15380-5. PMID: 15492217
Rosenthal LP, & Bodley JW. (1987) Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu. The Journal of biological chemistry, 262(23), 10955-9. PMID: 3301847
Chiron S, Suleau A, & Bonnefoy N. (2005) Mitochondrial translation: elongation factor tu is essential in fission yeast and depends on an exchange factor conserved in humans but not in budding yeast. Genetics, 169(4), 1891-901. PMID: 15695360
Ozturk SB, & Kinzy TG. (2008) Guanine nucleotide exchange factor independence of the G-protein eEF1A through novel mutant forms and biochemical properties. The Journal of biological chemistry, 283(34), 23244-53. PMID: 18562321
- January 3, 2011
- 12:03 PM
- 996 views
stringent response... in drosophila?
by Vasili Hauryliuk in stringent response
If you happened to be a bacteria, you must prepare for trouble: shortage of food, temperature changes and so on. And when the trouble comes, you should respond accordingly. Stringent response system does exactly that: it integrates several input sources (aminoacid, fat and carbon limitation, temperature upshift to name a few) and alters concentration of the alarmone molecule ppGpp (GDP with 2 extra phosphates attached to the sugar moiety).ppGpp in turn regulates everything: transcription, translation, replication. Therefore it is a must to control ppGpp levels very precisely, and to that end bacteria have proteins which produce ppGpp (RelA), (mostly)degrade it (SpoT) and do both (Rel). All together these proteins are called RelA-SpoT homologues (RSH).All this is in bacteria... how about eukaryotes? Well, organelles have RSH, with some of them being rather peculiar in terms of signals they respond to, such as Ca++ sensitive OsCRSH1 RSH from chloroplasts. But what about cytozolic proteins?Exactly that was discovered in 2010 - cytozolic RSH Mesh1 in drosophila. It is a small protein, capable of only ppGpp hydrolysis, which it does specifically: it takes only this nucleotide among the variety of different ones tested. Knock-out has a strong and specific phenotype, suggesting that the Mesh1 is important and functional. Moreover, Mesh1 can substitute SpoT in bacteria. X-ray structure of the protein was solved, so the paper went into Nature Structural Biology.The big question here is how did Mech1 end up in eukaryotes and why is it retained there? How do you transfer such a complex system as stringent response from bacteria to eukaryotes?With RSH proteins one can not just have a ppGpp-producing one - in this case you produce ppGpp, it accumulates and the bug dies. Therefore usually bugs have Rel - it both makes and degrades ppGpp, so the whole system is in one protein. Mesh1 degrades ppGpp, and no cytozolic RSH capable of synthesis were identified in eukaryotes so far... so what is Mesh1 doing? what is it degrading? May be there is some ppGpp-producing protein which is not homologous to RSH, and this s the source of ppGpp which Mesh1 degrades. Or may be Mesh1 actually does something else then ppGpp hydrolysis.It would be interesting to see some work done on determination of the ppGpp concentration in eukaryotic cells under different conditions, similarly to what is done for bacteria. Bacterial contamination might be a problem... But still, worth a try!References:Sun D, Lee G, Lee JH, Kim HY, Rhee HW, Park SY, Kim KJ, Kim Y, Kim BY, Hong JI, Park C, Choy HE, Kim JH, Jeon YH, & Chung J (2010). A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses. Nature structural & molecular biology, 17 (10), 1188-94 PMID: 20818390Mitkevich VA, Ermakov A, Kulikova AA, Tankov S, Shyp V, Soosaar A, Tenson T, Makarov AA, Ehrenberg M, & Hauryliuk V (2010). Thermodynamic characterization of ppGpp binding to EF-G or IF2 and of initiator tRNA binding to free IF2 in the presence of GDP, GTP, or ppGpp. Journal of molecular biology, 402 (5), 838-46 PMID: 20713063Maciag M, Kochanowska M, Lyzeń R, Wegrzyn G, & Szalewska-Pałasz A (2010). ppGpp inhibits the activity of Escherichia coli DnaG primase. Plasmid, 63 (1), 61-7 PMID: 19945481Gralla JD (2005). Escherichia coli ribosomal RNA transcription: regulatory roles for ppGpp, NTPs, architectural proteins and a polymerase-binding protein. Molecular microbiology, 55 (4), 973-7 PMID: 15686546Buckstein MH, He J, & Rubin H (2008). Characterization of nucleotide pools as a function of physiological state in Escherichia coli. Journal of bacteriology, 190 (2), 718-26 PMID: 17965154... Read more »
Sun D, Lee G, Lee JH, Kim HY, Rhee HW, Park SY, Kim KJ, Kim Y, Kim BY, Hong JI.... (2010) A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses. Nature structural , 17(10), 1188-94. PMID: 20818390
Mitkevich VA, Ermakov A, Kulikova AA, Tankov S, Shyp V, Soosaar A, Tenson T, Makarov AA, Ehrenberg M, & Hauryliuk V. (2010) Thermodynamic characterization of ppGpp binding to EF-G or IF2 and of initiator tRNA binding to free IF2 in the presence of GDP, GTP, or ppGpp. Journal of molecular biology, 402(5), 838-46. PMID: 20713063
Maciag M, Kochanowska M, Lyzeń R, Wegrzyn G, & Szalewska-Pałasz A. (2010) ppGpp inhibits the activity of Escherichia coli DnaG primase. Plasmid, 63(1), 61-7. PMID: 19945481
Gralla JD. (2005) Escherichia coli ribosomal RNA transcription: regulatory roles for ppGpp, NTPs, architectural proteins and a polymerase-binding protein. Molecular microbiology, 55(4), 973-7. PMID: 15686546
Buckstein MH, He J, & Rubin H. (2008) Characterization of nucleotide pools as a function of physiological state in Escherichia coli. Journal of bacteriology, 190(2), 718-26. PMID: 17965154
- December 30, 2010
- 11:19 AM
- 1,092 views
specific inhibition of nonsense-mediated mRNA decay by small molecule
by Vasili Hauryliuk in stringent response
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! 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: 17450125Manuvakhova 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... Read more »
Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P, Paushkin S, Patel M, Trotta CR, Hwang S.... (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
- December 27, 2010
- 08:48 AM
- 1,104 views
Double life of bacterial elongation factor EF-Tu
by Vasili Hauryliuk in stringent response
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: 16877446Defeu 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: 20133608Vats 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... Read more »
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
- December 18, 2010
- 07:59 AM
- 664 views
paper of the week: Kuntz et al., PNAS '99
by Vasili Hauryliuk in stringent response
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 (deltaG) 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... Read more »
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
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