Expression of a streptomycete leaderless mRNA encoding chloramphenicol acetyltransferase in Escherichia coli.
The chloramphenicol acetyltransferase (cat) gene from Streptomyces acrimycini encodes a leaderless mRNA. Expression of the cat coding sequence as a leaderless mRNA from a modified lac promoter resulted in chloramphenicol resistance in Escherichia coli. Transcript mapping with nuclease S1 confirmed that the 5' end of the cat message initiated at the A of the AUG translational start codon. Site-directed mutagenesis of the lac promoter or the cat start codon abolished chloramphenicol resistance, indicating that E. coli initiated translation at the 5' terminal AUG of the cat leaderless mRNA. Addition of 5'-AUGC-3' to the 5' end of the cat mRNA resulted in translation occurring also from the reading frame defined by the added AUG triplet, suggesting that a 5'-terminal start codon is an important recognition feature for initiation and establishing reading frame during translation of leaderless mRNA. Addition of an untranslated leader and Shine-Dalgarno sequence to the cat coding sequence increased cat expression in a cat:lacZ fusion; however, the level of expression was significantly lower than when a fragment of the bacteriophage lambda cI gene, also encoding a leaderless mRNA, was fused to lacZ. These results indicate that in the absence of an untranslated leader and Shine-Dalgarno sequence, the streptomycete cat mRNA is translated by E. coli; however, the cat translation signals, or other features of the cat mRNA, provide for only a low level of expression in E. coli.
Wu
C J
CJ
Department of Microbiology, Miami University, Oxford, Ohio 45056, USA.
Janssen
G R
GR
D002701:Chloramphenicol / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D002702:Chloramphenicol Resistance / Q000235:genetics; D018387:Codon, Initiator; D004926:Escherichia coli / Q000235:genetics; D007763:Lac Operon; D009154:Mutation; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012313:RNA / Q000096:biosynthesis; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*
D018387:Codon, Initiator; D012329:RNA, Bacterial; D012333:RNA, Messenger; C039919:RNA, recombinant; D011993:Recombinant Fusion Proteins; D012313:RNA; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase