Ivan Lomakin
Center for Structural Biology
Department of Molecular Biophysics & Biochemistry
Yale University
Structural studies of initiation of protein synthesis in eukaryotes
Summary:
The atomic structures of the small 30S ribosomal subunit from the thermophile bacterium Thermus thermophilus (1) and the large 50S ribosomal subunit from the halophile archaebacterium Haloarcula marismortui (2) provided enormous impact on our understanding of the general mechanism of the protein synthesis. This mechanism is most likely the same between prokaryotic and eukaryotic organisms due to the evolutionary conservation of rRNA and ribosomal proteins. However, eukaryotic rRNA is larger and each ribosomal subunit has more proteins then its prokaryotic counterpart. These structural differences reflect more complicated mechanism of ekaryotic translation. They are especially pronounced at the initiation stage. Translation initiation in eukariotes requires more then 12 protein factors (only 3 in prokaryotes) and utilizes the “cap” structure on the 5’-end of mRNA (3). To start protein synthesis small 40S ribosomal subunit scan 5’- untranslated region of mRNA to find initiation AUG codon – process that is unique for eukaryotic organisms (4). We are using X-ray crystallography to find how these functional differences correlate with structural properties of eukaryotic ribosomes.
Papers relating to this work:
1. Wimberly BT, Brodersen DE, Clemons WM Jr, Morgan-Warren RJ, Carter AP, Vonrhein C, Hartsch T, Ramakrishnan V. (2000)
Nature 407, 327-339
2. Ban N, Nissen P, Hansen J, Moore PB, Steitz TA. (2000)
Science 289, 905-920
3. Kapp LD, Lorsch JR. (2004)
Annu Rev Biochem. 73, 657-704.
4. Kozak M. (2002)
Gene 299(1-2), 1-34.
Structure of yeast fatty acid synthase
We have now determined the atomic structure of the yeast Saccharomyces cerevisiae fatty acid synthase (FAS) derived from two crystal forms of the enzyme that were obtained by a fortuitous accident. While attempting to crystallize the yeast 40S ribosomal subunit, we obtained instead these crystals of FAS, which cosediments at 40S with the small ribosomal subunit.
Summary:
In yeast, the whole metabolic pathway for making 16- and 18-carbon fatty acids is carried out by fatty acid synthase, a 2.6 megadalton molecular-
weight macromolecular assembly containing six copies of all eight catalytic centers. Wehave determined its crystal structure, which illuminates how this enzyme is initially activated and then carries out multiple steps of synthesis in each of six sterically isolated reaction chambers. Six of the catalytic sites are in the wall of the assembly facing an acyl carrier protein (ACP) bound to the ketoacyl synthase domain. Two-dimensional diffusion of substrates to the catalytic sites may be achieved by the electrostatically negative ACP swinging to each of the six electrostatically positive catalytic sites. The phosphopantetheinyl transferase domain lies outside the shell of the assembly, inaccessible to ACP that lies inside, suggesting that the attachment of the pantetheine arm to ACP must occur before complete assembly of the complex.
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Publications:
1. Lomakin IB*, Steitz, TA*
The initiation of mammalian protein synthesis and mRNA scanning mechanism.
Nature,2013 500:307-311.*-corresponding author
2. Lomakin IB, Xiong Y, Steitz TA.
The crystal structure of yeast fatty acid synthase, a cellular machine with eight active sites working together.
Cell. 2007 Apr 20;129(2):319-32.
3. Unbehaun A, Marintchev A, Lomakin IB, Didenko T, Wagner G, Hellen CU, Pestova TV.
Position of eukaryotic initiation factor eIF5B on the 80S ribosome mapped by directed hydroxyl radical probing.
EMBO J. 2007 Jun 14; [Epub ahead of print]
4. Lomakin IB, Shirokikh NE, Yusupov MM, Hellen CU, Pestova TV.
The fidelity of translation initiation: reciprocal activities of eIF1, IF3 and YciH.
EMBO J. 2006 Jan 11;25(1):196-210.
5. Kolupaeva VG, Unbehaun A, Lomakin IB, Hellen CU, Pestova TV.
Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association.
RNA. 2005 Apr;11(4):470-86.
6. Pestova TV, Lomakin IB, Hellen CU.
Position of the CrPV IRES on the 40S subunit and factor dependence of IRES/80S ribosome assembly.
EMBO Rep. 2004 Sep;5(9):906-13.
7. Laptenko O, Lee J, Lomakin I, Borukhov S.
Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase.
EMBO J. 2003 Dec 1;22(23):6322-34.
8. Lomakin IB, Kolupaeva VG, Marintchev A, Wagner G, Pestova TV.
Position of eukaryotic initiation factor eIF1 on the 40S ribosomal subunit determined by directed hydroxyl radical probing.
Genes Dev. 2003 Nov 15;17(22):2786-97.
9. Kolupaeva VG*, Lomakin IB*, Pestova TV, Hellen CU.
Eukaryotic initiation factors 4G and 4A mediate conformational changes downstream of the initiation codon of the encephalomyocarditis virus internal ribosomal
entry site.
Mol Cell Biol. 2003 Jan;23(2):687-98.
*V.G.K. and I.B.L. contributed equally to this work.
10. Wang H, Iacoangeli A, Popp S, Muslimov IA, Imataka H, Sonenberg N, Lomakin IB, Tiedge H.
Dendritic BC1 RNA: functional role in regulation of translation initiation.
J Neurosci. 2002 Dec 1;22(23):10232-41.
11. Pestova TV, Kolupaeva VG, Lomakin IB, Pilipenko EV, Shatsky IN, Agol VI, Hellen CU.
Molecular mechanisms of translation initiation in eukaryotes.
Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7029-36. Review.
12. Marcotrigiano J, Lomakin IB, Sonenberg N, Pestova TV, Hellen CU, Burley SK.
A conserved HEAT domain within eIF4G directs assembly of the translation initiation machinery.
Mol Cell. 2001 Jan;7(1):193-203.
13. Lomakin IB, Hellen CU, Pestova TV.
Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of
encephalomyocarditis virus and is required for internal initiation of translation.
Mol Cell Biol. 2000 Aug;20(16):6019-29.
14. Pestova TV, Lomakin IB, Lee JH, Choi SK, Dever TE, Hellen CU.
The joining of ribosomal subunits in eukaryotes requires eIF5B.
Nature. 2000 Jan 20;403(6767):332-5.
15. Kulish D, Lee J, Lomakin I, Nowicka B, Das A, Darst S, Normet K, Borukhov S.
The functional role of basic patch, a structural element of Escherichia coli transcript cleavage factors GreA and GreB.
J Biol Chem. 2000 Apr 28;275(17):12789-98.
16. Lomakin IB, Mashko SV, Epishin SM, Ketlinskii SA, Konusova VG, Vinetskii IuP, Debabov VG.
Biosynthesis of human recombinant interleukin-8 in Escherichia coli.
Dokl Akad Nauk. (Proc Natl Acad Sci Russia) 1993 Feb;328(4):513-6. Russian.
17. Lebedeva MI, Tsuba NA, Kotenko SV, Epishin SM, Lomakin IB, Vinetski YuP, Mironov AA, Ketlinsky SA, Mashko SV.
Efficient Expression of Human Interleukin-1Gene in Escherichia coli under the Control of a T7 RNA Polymerase derived System.
Biotechnologiya 1993, 4: 18. Russian
18. Lomakin IB, Lebedeva MI, Konusova VG, Vilichko EV, Lebedev VF, Vinetski YuP, Ketlinsky SA, Mashko SV.
T7 RNA Polymerase derived Expression of the Human Interleukin-8 cDNA in Escherichia coli. Purification and Initial Characterisation of Recombinant hIL-8. Biotechnologiya 1993, 10, 10-15. Russian
19. Prokopenko IV, Lomakin IB, Zakharova ES, Vinetskii IuP.
Cloning preprolactin cDNA from Pacific salmon in Escherichia coli.
Mol Biol (Mosk). 1991 May-Jun;25(3):689-94. Russian.
20. Kotenko SV, Bulenkov MT, Veiko VP, Epishin SM, Lomakin IB, Emel’ianov AV, Kozlov AP, Konusova VG, Kotov AIu, Kurbatova TV, Reshetnikov VL, Simbircev AS, Ketlinsky SA, Vinetski YuP.
Cloning and structural analysis of cDNA coding for human prointerleukin-1 alpha and prointerleukin-1 beta.
Mol Gen Mikrobiol Virusol. 1989 Dec;(12):13-7. Russian.
21. Kotenko SV, Bulenkov MT, Veiko VP, Epishin SM, Lomakin IB, Emelyanov AV, Kozlov AP, Konusova VG, Kotov AY, Kurbatova TV, Reshetnikov VL, Simbircev AS, Ketlinsky SA, Vinetski YuP.
Cloning of the cDNA coding for human prointerleukin-1 alpha and prointerleukin-1 beta.
Dokl Akad Nauk SSSR. (Proc Natl Acad Sci USSR) 1989;309(4):1005-8. Russian.
22. Zakharova ES, Kotenko SV, Lomakin IB, Epishin SM, Vinetskii IuP.
Nucleotide sequence of cDNA of glicinin A3 subunits: variability of different species detected by comparative analysis.
Genetika. 1986 Dec;22(12):2741-9. Russian.