Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 124-8 |
| Number of pages | 5 |
| Journal | Biochimica et Biophysica Acta |
| Volume | 1050 |
| Issue number | 1-3 |
| Publication status | Published - 27 Aug 1990 |
Keywords
- Animals
- Base Sequence
- Chloramphenicol O-Acetyltransferase
- Cloning, Molecular
- Encephalomyocarditis virus
- Models, Molecular
- Mutagenesis, Site-Directed
- Nucleic Acid Conformation
- Peptide Chain Initiation, Translational
- Plasmids
- Protein Biosynthesis
- RNA, Messenger
- RNA, Viral
- Restriction Mapping
- Reticulocytes
- Trans-Activators
- Transcription, Genetic