Mechanism of selenoprotein synthesis in bacteria
Final Report Abstract
In bacteria, the recoding of a UGA stop codon for selenocysteine requires a specialized translation factor SelB, a non-canonical Sec-tRNASec, and a selenocysteine incorporation sequence (SECIS) in the mRNA. The present project was aimed at understanding the molecular mechanisms of selenocysteine insertion into proteins. The efficiency of Sec insertion in vivo was measured using dual-reporter Sec insertion assay. We found that at conditions where the concentration of external selenium was not limiting, the efficiency of UGA recoding by Sec was close to 40% regardless of the growth rate of the cells. RF2 competed with SelB•GTP•Sec-tRNASec delivery to the ribosome in a codon-dependent fashion. However, given that RF2 is present in the cell inlarge excess over the components of the Sec-insertion machinery, SelB must have a mechanism to escape a direct competition for the UGA codon. This is achieved by the early recruitment of SelB to the SECIS during the translation of mRNA region preceding the UGA stop codon, which inhibits the entry of RF2 into the decoding site of the ribosome. The kinetic mechanism of the interaction of the ternary complex SelB•GTP•Sec-tRNASec with the ribosome was studied using a combination of biochemical and biophysical techniques. Sec-tRNASec binds to SelB•GTP with an extraordinary high affinity (Kd = 0.2 pM). The tight binding is driven enthalpically and involves the net formation of four ion pairs, three of which may involve the Sec residue. Thermodynamic coupling in binding of Sec-tRNASec and GTP to SelB ensures at the same time the specificity of Sec- versus Ser-tRNASec selection and rapid release of Sec-tRNASec from SelB after GTP cleavage on the ribosome.SelB•GTP•Sec-tRNASec complex is recruited to the ribosome very rapidly. Codon recognition engages Sec-tRNA in a tight complex with the ribosome. The following GTP hydrolysis in SelB is delayed and is rate-limiting for the Sec incorporation into peptides. SelB provides an example for the evolution of a highly specialized protein-RNA complex towards recognition of unique set of identity elements. The unusually high affinity of SelB to Sec-tRNASec is required to protect utterly labile Sec-tRNASec from destruction by hydrolysis or oxidation and deliver it to the ribosome. After GTP hydrolysis,SelB changes its conformation to increase the dissociation rate of Sec-tRNASec by six orders of magnitude, which becomes sufficiently high to allow for rapid transfer of tRNA from the factor to the ribosome. Thus, the conformational rearrangements of SelB are crucial for Sec delivery and incorporation on the ribosome translating mRNAs coding for selenoproteins.
Publications
- Recognition of selenocysteyl-tRNASec by elongation factor SelB. PhD thesis, Universität Witten/Herdecke (2009)
Palesskava, A.
- (2010) Thermodynamic and kinetic framework of selenocysteyl-tRNASec recognition by elongation factor SelB. J. Biol. Chem. 285, 3014-3020
Paleskava, A., Konevega, A. L., Rodnina, M.V.
- Molecular mechanism of selenocysteine incorporation in bacterial translation. PhD thesis,Georg-August-Universität Göttingen (2011)
Kotini, S.B.
- (2012) Thermodynamics of the GTP-GDP operated conformational switch of slenocysteine-specific translation factor SelB. J. Biol.Chem.287, 27906-27012
Paleskava A., Konevega A.L., Rodnina M.V.
(See online at https://doi.org/10.1074/jbc.M112.366120)
