Towards understanding selenocysteine incorporation into bacterial proteins.
In bacteria, UGA stop codons can be recoded to direct the incorporation of selenocysteine into proteins 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-tRNASec, which is formed from Ser-tRNASec by selenocysteine synthase, SelA, using selenophosphate as selenium donor. Here we describe a rapid-kinetics approach to study the mechanism of selenocysteine insertion into proteins on the ribosome. Labeling of SelB, Sec-tRNASec and other components of the translational machinery allows direct observation of the formation or dissociation of complexes by monitoring changes in the fluorescence of single dyes or fluorescence resonance energy transfer 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 that has a ring-like structure with five bilobed wings, similar to the structure of the E. coli complex determined previously.
Fischer
Niels
N
3D Electron Cryomicroscopy Group, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
Paleskava
Alena
A
Gromadski
Kirill B
KB
Konevega
Andrey L
AL
Wahl
Markus C
MC
Stark
Holger
H
Rodnina
Marina V
MV
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020285:Cryoelectron Microscopy; D007700:Kinetics; D008954:Models, Biological; D012345:RNA, Transfer, Amino Acid-Specific / Q000737:chemistry / Q000378:metabolism; D017279:Selenocysteine / Q000737:chemistry / Q000378:metabolism*; D046968:Thermoanaerobacter / Q000201:enzymology / Q000378:metabolism; D014166:Transferases / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*
D001426:Bacterial Proteins; D012345:RNA, Transfer, Amino Acid-Specific; C036928:tRNA, selenocysteine-; D017279:Selenocysteine; D014166:Transferases; C060600:selenium transferase