Thermodynamic and Kinetic Framework of Selenocysteyl-tRNASec Recognition by Elongation Factor SelB

Abstract

Sec selection and rapid release of Sec-tRNA Sec from SelB after GTP cleavage on the ribosome. SelB provides an example for the evolution of a highly specialized protein-RNA complex toward recognition of unique set of identity elements. The mode of tRNA recognition by SelB is reminiscent of another specialized factor, eIF2, rather than of EF-Tu, the common delivery factor for all other aminoacyl-tRNAs, in line with a common evolutionary ancestry of SelB and eIF2.

The pathway to GTPase activation of elongation factor SelB on the ribosome

“…(IF1, IF2, IF3), and fMet-tRNA fMet were prepared as described [47][48][49] . E. coli SelA and SelD (gift from M. Wahl, Free University of Berlin) and SelB 50 containing a hexahistidine tag (gift from A. Böck, LMU Munich) were expressed in BL21(DE3) cells and purified according to published protocols 4,51 . Biochemical analysis showed that SelB carrying the His-tag is fully functional in the interactions with guanine nucleotides, SECIS elements, and Sec-tRNA Sec and in mediating UGA recoding in vivo 4,50 .…”

“…E. coli SelA and SelD (gift from M. Wahl, Free University of Berlin) and SelB 50 containing a hexahistidine tag (gift from A. Böck, LMU Munich) were expressed in BL21(DE3) cells and purified according to published protocols 4,51 . Biochemical analysis showed that SelB carrying the His-tag is fully functional in the interactions with guanine nucleotides, SECIS elements, and Sec-tRNA Sec and in mediating UGA recoding in vivo 4,50 . tRNA Sec was overexpressed in E. coli BL21 transformed with the plasmid pCB2013 (gift from A. Böck, LMU Munich) 52 and purified and aminoacylated as described 4,53,54 .…”

“…Biochemical analysis showed that SelB carrying the His-tag is fully functional in the interactions with guanine nucleotides, SECIS elements, and Sec-tRNA Sec and in mediating UGA recoding in vivo 4,50 . tRNA Sec was overexpressed in E. coli BL21 transformed with the plasmid pCB2013 (gift from A. Böck, LMU Munich) 52 and purified and aminoacylated as described 4,53,54 . The mRNA UGA was a derivative of mLP75s 54 (Extended Data Fig.…”

“…Complex preparation for cryo-EM. 70S ribosomes (3 μ M) were incubated with IF1, IF2, IF3 (4.5 μ M), mRNA (15 μ M), and f[ 3 H]Met-tRNA fMet (7 μ M) in buffer A (50 mM Tris-HCl (pH 7.5), 70 mM NH 4 Cl, 30 mM KCl, 7 mM MgCl 2 , 2 mM DTT) with 1 mM GTP for 30 min at 37 °C. Initiation efficiency was close to 100% as verified by nitrocellulose binding.…”

“…These include the universal elongation factor EF-Tu (in bacteria, or eEF1A in eukaryotes) and a specialized factor SelB 1 (eEFSec-SBP2 in eukaryotes 2 ). EF-Tu and SelB deliver aminoacyl-tRNA (aa-tRNA) to the ribosome at the cost of GTP hydrolysis 3,4 . In contrast to EF-Tu, which is a carrier for all canonical aa-tRNAs, SelB is specialized to bind only the tRNA that is specific for Sec 5,6 .…”