Functional analysis of the interplay between translation termination, selenocysteine codon context, and selenocysteine insertion sequence-binding protein 2.
A selenocysteine insertion sequence (SECIS) element in the 3'-untranslated region and an in-frame UGA codon are the requisite cis-acting elements for the incorporation of selenocysteine into selenoproteins. Equally important are the trans-acting factors SBP2, Sec-tRNA[Ser]Sec, and eEFSec. Multiple in-frame UGAs and two SECIS elements make the mRNA encoding selenoprotein P (Sel P) unique. To study the role of codon context in determining the efficiency of UGA readthrough at each of the 10 rat Sel P Sec codons, we individually cloned 27-nucleotide-long fragments representing each UGA codon context into a luciferase reporter construct harboring both Sel P SECIS elements. Significant differences, spanning an 8-fold range of UGA readthrough efficiency, were observed, but these differences were dramatically reduced in the presence of excess SBP2. Mutational analysis of the "fourth base" of contexts 1 and 5 revealed that only the latter followed the established rules for hierarchy of translation termination. In addition, mutations in either or both of the Sel P SECIS elements resulted in differential effects on UGA readthrough. Interestingly, even when both SECIS elements harbored a mutation of the core region required for Sec incorporation, context 5 retained a significantly higher level of readthrough than context 1. We also show that SBP2-dependent Sec incorporation is able to repress G418-induced UGA readthrough as well as eRF1-induced stimulation of termination. We conclude that a large codon context forms a cis-element that works together with Sec incorporation factors to determine readthrough efficiency.
Gupta
Malavika
M
Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Copeland
Paul R
PR
D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002474:Cell-Free System / Q000378:metabolism; D003049:Coccidiostats / Q000494:pharmacology; D018388:Codon, Terminator / Q000235:genetics / Q000378:metabolism*; D005839:Gentamicins / Q000494:pharmacology; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D010443:Peptide Chain Termination, Translational / Q000187:drug effects / Q000502:physiology*; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017279:Selenocysteine / Q000235:genetics / Q000378:metabolism*; D051149:Selenoprotein P / Q000096:biosynthesis / Q000235:genetics
D020413:3' Untranslated Regions; D003049:Coccidiostats; D018388:Codon, Terminator; D005839:Gentamicins; D010445:Peptide Elongation Factors; D010454:Peptide Termination Factors; D012346:RNA, Transfer, Amino Acyl; D016601:RNA-Binding Proteins; D051149:Selenoprotein P; C119984:selenocysteine insertion sequence binding protein, mammalian; C112705:selenocysteinyl-tRNA; D017279:Selenocysteine; C010680:antibiotic G 418; D008156:Luciferases