The ultimate aim of the project is to understand the structural rearrangements of the ribosome that underlie the different phases of protein synthesis in bacteria and yeast. To achieve this goal, functional and structural analyses will be fully integrated to maximize synergisms between the different competence areas of the participating groups. In detail, the project aims ? ? (1) To clarify how initiation of protein synthesis takes place with the help of the three initiation factors IF1, IF2 (a G protein) and IF3. ? (2) To study how translocation of tRNAs occurs on the eubacterial ribosome with the help of elongation factor EF-G (a G protein). ? (3) To study the solution structure of eubacterial class-1 release factors to decide if it is similar to the crystal structure of RF2. ? (4) To clarify the role of domain 1 in eubacterial release factors and the role of protein L11 in the large ribosomal subunit in termination of protein synthesis. ? (5) To clarify the mechanism behind ribosomal recycling, catalysed by EF-G and ribosomal recycling factor RRF, back to a new round of initiation. ? (6) To clarify the role of peptide release factor eRF3 (a G protein) in eukaryotic protein synthesis. ? ? All experiments are based on in vitro systems with components of high purity from E. coli and yeast. The approach is to integrate functional assay carried out with biochemical tools, including fast kinetics methods (quench-flow, stopped flow) with structural analysis carried out mainly with cryo-electron microscopy but also with low angle X-ray scattering. Ribosomal complexes will be shipped from Uppsala to Albany and prepared under controlled temperature conditions for cryo-EM studies, including time resolved cryo-EM when applicable. Grids will be imaged with a liquid nitrogen or liquid helium cooled cryo-EM microscope, and the images will be analyzed with the SPIDER image processing system. Between 30,000 and 100,000 images will be collected for 10-12A resolution. The density maps will be analyzed by manual or automated fitting of x-ray structures or by real space refinement to obtain atomic models. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070768-04
Application #
7229012
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Flicker, Paula F
Project Start
2004-05-01
Project End
2009-04-30
Budget Start
2007-05-01
Budget End
2009-04-30
Support Year
4
Fiscal Year
2007
Total Cost
$199,364
Indirect Cost
Name
Uppsala University
Department
Type
DUNS #
350582201
City
Uppsala
State
Country
Sweden
Zip Code
SE-75-1 05
Mitkevich, Vladimir A; Ermakov, Andrey; Kulikova, Alexandra A et al. (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. J Mol Biol 402:838-46
Villa, Elizabeth; Sengupta, Jayati; Trabuco, Leonardo G et al. (2009) Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis. Proc Natl Acad Sci U S A 106:1063-8
Hauryliuk, Vasili; Mitkevich, Vladimir A; Draycheva, Albena et al. (2009) Thermodynamics of GTP and GDP binding to bacterial initiation factor 2 suggests two types of structural transitions. J Mol Biol 394:621-6
Lovmar, Martin; Nilsson, Karin; Lukk, Eliisa et al. (2009) Erythromycin resistance by L4/L22 mutations and resistance masking by drug efflux pump deficiency. EMBO J 28:736-44
Li, Wen; Agirrezabala, Xabier; Lei, Jianlin et al. (2008) Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM. EMBO J 27:3322-31
Johansson, Magnus; Lovmar, Martin; Ehrenberg, Mans (2008) Rate and accuracy of bacterial protein synthesis revisited. Curr Opin Microbiol 11:141-7
Hauryliuk, Vasili; Hansson, Sebastian; Ehrenberg, Mans (2008) Cofactor dependent conformational switching of GTPases. Biophys J 95:1704-15
Hauryliuk, Vasili; Mitkevich, Vladimir A; Eliseeva, Natalia A et al. (2008) The pretranslocation ribosome is targeted by GTP-bound EF-G in partially activated form. Proc Natl Acad Sci U S A 105:15678-83
Gao, Ning; Zavialov, Andrey V; Ehrenberg, Mans et al. (2007) Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits. J Mol Biol 374:1345-58
Allen, Gregory S; Frank, Joachim (2007) Structural insights on the translation initiation complex: ghosts of a universal initiation complex. Mol Microbiol 63:941-50

Showing the most recent 10 out of 13 publications