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Role of Cis Expression and Translational Coupling in the Assembly of a Functional Drug Pump
Author(s) -
Pradhan Prajakta,
Kaur Parjit
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a517-a
Subject(s) - drug , coupling (piping) , posttranslational modification , expression (computer science) , protein expression , chemistry , pharmacology , medicine , computer science , biochemistry , engineering , mechanical engineering , gene , programming language , enzyme
DrrAB proteins confer resistance to the anticancer drug, doxorubicin, in Streptomyces peucetius . Since the DrrAB efflux pump is homologous to P‐glycoprotein (P‐gp), the DrrAB system is expected to serve as a good model to understand the assembly and function of Pgp. DrrA, the ATP binding subunit, and DrrB, the transmembrane subunit, have been previously shown to be biochemically coupled. Furthermore, drr A and drr B genes in the wild type operon have overlapping stop and start codons (ATGA) indicating translational coupling between the two genes. In the wild type cis system, the DrrA and DrrB proteins form a functional complex and impart resistance to the drug doxorubicin. Although the expression of DrrB can be rescued by expression of DrrA in trans the functional phenotype of doxorubicin resistance cannot be achieved. The objective of this study was to understand the role of cis expression and translational coupling in the assembly of the DrrAB complex. An early stop codon was introduced in the upstream gene drr A to bring about premature termination of translation of DrrA. Expression analysis indicated that expression of DrrB is translationally coupled to the upstream ORF. In order to investigate the importance of drr A in cis location, drr A gene was substituted with an unrelated gene referred to here as gene X. Expression analysis indicated that translation of drr B could be achieved when coupled to any upstream ORF. Functional assays in the near future will show if translational coupling may be sufficient for formation of a functional complex. This study is expected to elucidate the role of different factors in successful assembly of the DrrAB drug efflux pump. It is likely that the key to assembly of DrrAB lies in co‐folding of the two protein components, which can be accomplished only by maintaining the genes in cis in a translationally coupled manner. RO1GM51981‐07