Two distinct SECIS structures capable of directing selenocysteine incorporation in eukaryotes.
Translation of UGA as selenocysteine requires specific RNA secondary structures in the mRNAs of selenoproteins. These elements differ in sequence, structure, and location in the mRNA, that is, coding versus 3' untranslated region, in prokaryotes, eukaryotes, and archaea. Analyses of eukaryotic selenocysteine insertion sequence (SECIS) elements via computer folding programs, mutagenesis studies, and chemical and enzymatic probing has led to the derivation of a predicted consensus structural model for these elements. This model consists of a stem-loop or hairpin, with conserved nucleotides in the loop and in a non-Watson-Crick motif at the base of the stem. However, the sequences of a number of SECIS elements predict that they would diverge from the consensus structure in the loop region. Using site-directed mutagenesis to introduce mutations predicted to either disrupt or restore structure, or to manipulate loop size or stem length, we show that eukaryotic SECIS elements fall into two distinct classes, termed forms 1 and 2. Form 2 elements have additional secondary structures not present in form 1 elements. By either insertion or deletion of the sequences and structures distinguishing the two classes of elements while maintaining appropriate loop size, conversion of a form 1 element to a functional form 2-like element and of a form 2 to a functional form 1-like element was achieved. These results suggest commonality of function of the two classes. The information obtained regarding the existence of two classes of SECIS elements and the tolerances for manipulations of stem length and loop size should facilitate designing RNA molecules for obtaining high-resolution structural information about these elements.
Grundner-Culemann
E
E
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Martin
G W
GW
Harney
J W
JW
Berry
M J
MJ
D000818:Animals; D001105:Archaea / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003062:Codon / Q000235:genetics*; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics; D004285:Dogs; D005057:Eukaryotic Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D007668:Kidney; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism; D011387:Prokaryotic Cells / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D017279:Selenocysteine / Q000378:metabolism*; D051140:Selenoproteins; D014162:Transfection
D003062:Codon; D009841:Oligonucleotides; D011506:Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D051140:Selenoproteins; D017279:Selenocysteine; D004247:DNA