Downstream sequence adjacent to AUG affects translation of chloramphenicolacetyl transferase in eukaryotic cells

The CAT gene is widely used as a reporter in eukaryotic systems because of the efficient translation of its mRNA. We report here that a sequence occur ring in the CAT mRNA at +15 nucleotides from CAT AUG is essential for trans lation. This sequence includes a stem-loop structure which, however, exhibi ts a calculated stability significantly lower than that required for a hair pin to act as an enhancer of translation in vitro. Replacement of this regi on with the corresponding sequence from mRNAs that are normally translated in eukaryotic systems drastically reduced translation of CAT in COS cells, although the consensus sequence around the AUG, known to be required for hi gh-level translation initiation, was conserved. These observations may be r elevant for the exploitation of the CAT reporter system for analysis of the mechanisms of translation initiation by means of fusion constructs.