Expression of a streptomycete leaderless mRNA encoding chloramphenicol acetyltransferase in Escherichia coli

Abstract

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.The translation frequency for procaryotic mRNA containing a 5Ј untranslated leader region is determined, in part, by the extent of complementarity between a Shine-Dalgarno (SD) sequence within the leader region and the anti-Shine-Dalgarno (ASD) sequence located near the 3Ј end of the 16S rRNA (8,9,28,30). Small subunit rRNAs from procaryotic ribosomes contain the conserved ASD region, and most procaryotic mRNAs contain a readily identifiable SD sequence. While the contributions of the SD-ASD interaction to translation are likely to be mechanistically similar among all procaryotes, other features of the translation initiation region have been proposed to also contribute to translation levels (6,18,21,29). The identification and analysis of mRNA features that influence translation levels are important for understanding the translation initiation process and for considerations of optimizing expression levels when genes are expressed within heterologous hosts.Although untranslated leader regions and SD sequences are found at the 5Ј ends of most procaryotic mRNAs, some genes encode leaderless mRNA whereby transcription and translation initiate at the same position. While genes that encode leaderless mRNA are relatively rare, more than 30 have been identified (11,23,32) since the Escherichia coli phage cI repressor was first reported in 1976 (22). Observations of leaderless mRNA (11) in Bacteria, Archaea, Eucarya, and eucaryotic organelles suggest that sequence and/or structural information contained within the coding sequence are sufficient to signal the translat...

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

“…PCR amplification with Pfu DNA polymerase (Stratagene), pIU1041 (Wu and Janssen, 1997) as template, a downstream primer (5Ј-ACGCTCATCGATAATTTCACC-GCC-3Ј), and each of the following upstream primers was used to prepare blunt-ended DNA fragments of cI-lacZ with the indicated 5Ј terminal start codons: AUG: 5Ј-ATGAGCA-CAAAAAAGAAACCATTAAC-3Ј; UAG: 5Ј-TAGAGCACAA-AAAAGAAACCATTAAC-3Ј; UAUAG: 5Ј-TATAGAGCACA-AAAAAGAAACCATTAAC-3Ј. The reaction products were cloned separately into a plasmid containing a lacUV5 promoter such that transcription was expected to initiate at the first position of the cloned fragment or a plasmid where the cloning site mapped to the translational start site of a modified leadered lacZ.…”

“…The widespread occurrence of leaderless mRNA suggests that translation of leaderless mRNA might represent a fundamental capability of all translation systems. In possible support of this, it has also been shown that leaderless mRNAs are translated in heterologous hosts (Janssen, 1993;Wu and Janssen, 1997).…”

Characterization of a two-component regulatory system from Acinetobacter baumannii that controls biofilm formation and cellular morphology

“…Total RNA was isolated from the parental 19606 strain and the Bfm17 and Bfm273 mutants as described previously (Wu & Janssen, 1997), and each RNA sample was further purified using the Qiagen RNeasy Total RNA Isolation System including the RNase-free DNase incolumn treatment, before amplification using one-step RT-PCR (Qiagen). The primers 1978 (59-TACGTGGTATCGAATACG-39) and2036 (59-ATTGCTTGCATCGTAACG-39) (Fig.…”

The siderophore-mediated iron acquisition systems of Acinetobacter baumannii ATCC 19606 and Vibrio anguillarum 775 are structurally and functionally related

“…The transcription of the bau genes was tested by RT-PCR analysis using total RNA isolated from bacteria grown under iron-limiting conditions as described before (Wu & Janssen, 1997). The RNA samples were treated with RNase-free DNase I (Roche) and used with an RT-PCR commercial kit (Qiagen) under the conditions suggested by the manufacturer.…”