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Dynamics and efficiency in vivo of UGA‐directed selenocysteine insertion at the ribosome
Author(s) -
Suppmann Sabine,
Persson Britt C.,
Böck August
Publication year - 1999
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/18.8.2284
Subject(s) - biology , selenocysteine , ribosome , genetics , transfer rna , translation (biology) , computational biology , microbiology and biotechnology , rna , messenger rna , biochemistry , gene , enzyme , cysteine
The kinetics and efficiency of decoding of the UGA of a bacterial selenoprotein mRNA with selenocysteine has been studied in vivo . A gst–lacZ fusion, with the fdhF SECIS element ligated between the two fusion partners, gave an efficiency of read‐through of 4–5%; overproduction of the selenocysteine insertion machinery increased it to 7–10%. This low efficiency is caused by termination at the UGA and not by translational barriers at the SECIS. When the selenocysteine UGA codon was replaced by UCA, and tRNA Sec with anticodon UGA was allowed to compete with seryl‐tRNA Ser1 for this codon, selenocysteine was found in 7% of the protein produced. When a non‐cognate SelB–tRNA Sec complex competed with EF‐Tu for a sense codon, no effects were seen, whereas a non‐cognate SelB–tRNA Sec competing with EF‐Tu‐mediated Su7‐tRNA nonsense suppression of UGA interfered strongly with suppression. The induction kinetics of β‐galactosidase synthesis from fdhF ′ – ′ lacZ gene fusions in the absence or presence of SelB and/or the SECIS element, showed that there was a translational pause in the fusion containing the SECIS when SelB was present. The results show that decoding of UGA is an inefficient process and that using the third dimension of the mRNA to accommodate an additional amino acid is accompanied by considerable quantitative and kinetic costs.