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A polylinker‐derived sequence, PL, highly increased translation efficiency in Escherichia coli
Author(s) -
Xu Jianhua,
Mironova Roumiana,
Ivanov Ivan G.,
Abouhaidar Mounir G.
Publication year - 1999
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/(sici)1521-4028(199903)39:1<51::aid-jobm51>3.0.co;2-0
Subject(s) - microbiology and biotechnology , biology , gene , messenger rna , ribosomal binding site , ribosome , gene expression , expression vector , translation (biology) , nucleotide , mutant , genetics , rna , recombinant dna
Pokeweed (Phytolacca americana) antiviral protein (PAP) is a highly specific ribosome‐inactivating glycosidase. The PAP gene was isolated and cloned in an expression vector containing a polylinker‐derived sequence (PL) but devoid of a Shine‐Dalgarno (SD) sequence. Surprisingly, E. coli cells transformed with this vector produced over twice the amount of PAP than that with the consensus SD sequence. Computer analysis of the 5′ terminal region of PAP mRNA revealed a nucleotide sequence (ACCUACUCGAGUUAG) which was complementary to two domains in 16S rRNA. The heptanucleotide ACCUACU (box I) is complementary to nucleotides 1434‐1440 and the GAGUUAG (box II) to nucleotides 507‐513 in 16S rRNA of E. coli . To examine the role of this sequence in the translation of PAP mRNA, single or both boxes were mutated and the protein yield was measured. Mutation of box I and of box II resulted in a 2.7 and 5.3 fold decrease in protein yield respectively, indicating that the PAP gene expression was dependent on the presence of both boxes. To investigate whether PL also increases expression of other genes, human calcitonin monomeric and tetrameric genes were used as reporters. It was found that the expression level was doubled compared to that by SD. These results demonstrate that the PL is an efficient translational initiator and may be used for high level expression of certain genes in E. coli . The possible mechanisms for the high level expression are discussed.