An initiation signal in the 5' untranslated leader sequence of encephalomyocarditis virus RNA.
A 593 nucleotide fragment of the 5' leader of encephalomyocarditis virus RNA (EMCV-RNA) was linked to the SP6 promoter and inserted upstream of the reporter gene chloramphenicol acetyltransferase (CAT). The presence of the 5'-UTR of EMCV-RNA in the RNA transcripts, made in vitro with the SP6 polymerase, resulted in a strong translational enhancement when tested in the micrococcal nuclease-treated reticulocyte lysate. The transcripts were equally active with or without a 5' methylated capstructure as expected, since EMCV-RNA is one of the mRNAs capable of internal initiation. We searched for a signal in the 5' leader that allows the 43S preinitiation complex to bind internally and localized a hairpin containing a unique nucleotide sequence, CUUUA, present in a domain conserved among cardio- and aphtoviral RNAs. Replacing this sequence into AGCU resulted in a 50% loss of translational activity. A second mutation involving a U-G change in the stem of that hairpin resulted in an almost complete loss of initiation.
Oudshoorn
P
P
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
Thomas
A
A
Scheper
G
G
Voorma
H O
HO
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D004686:Encephalomyocarditis virus / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic
D012333:RNA, Messenger; D012367:RNA, Viral; D015534:Trans-Activators; D015500:Chloramphenicol O-Acetyltransferase