You are here: Home :: Area NEM :: Life sciences :: Biochemistry

Niels Fischer, Alena Paleskava, Kirill B. Gromadski, Andrey L. Konevega, Markus C. Wahl, Holger Stark, Marina V. Rodnina

Towards understanding selenocysteine incorporation into bacterial proteins

Keywords: electron cryomicroscopy, Fluorescence, protein synthesis, recoding, ribosome, selenocysteine

In bacteria, UGA stop codons can be recoded to direct the incorporation of selenocysteine into protein on the ribosome. Recoding requires a selenocysteine incorporation sequence (SECIS) downstream of the UGA codon, a specialized translation factor SelB, and the non-canonical Sec-tRNA^Sec, which is formed from Ser-tRNA^Sec by selenocysteine synthase, SelA, using selenophosphate as donor of selenium. Here we describe a rapid-kinetics approach to study the mechanism of selenocysteine insertion into proteins on the ribosome. Labeling of SelB, SectRNA^Sec , and other components of the translational machinery allows for the direct observation of the formation or dissociation of complexes by monitoring changes in the fluorescence of single dyes or fluorescence resonance energy transfer (FRET) between two fluorophores. Furthermore, the structure of SelA was studied by electron cryomicroscopy (cryo-EM). We report that intact SelA from the thermophilic bacterium Moorella thermoacetica (mthSelA) can be vitrified for cryo-EM using a controlled-environment vitrification system. Two-dimensional image analysis of vitrified mthSelA images shows that SelA can adopt the wide range of orientations required for high resolution structure determination by cryo-EM. The results indicate that mthSelA forms a homodecamer which has a ring-like structure with five bi-lobed wings, similar to the structure of the E. coli complex determined previously.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 2007
Pages: -

Show full article (external site)

Show all available items of this journal

 

Area NEM