Date of Award

11-30-2008

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Parjit Kaur - Chair

Second Advisor

John Houghton

Third Advisor

Zehava Eichenbaum

Fourth Advisor

Phang Tai

Abstract

The DrrAB proteins of Streptomyces peucetius belong to the ABC family of ubiquitous membrane transporters. The DrrA and DrrB proteins together form a drug efflux pump that carries out the transport of the anticancer drug doxorubicin by carrying out ATP hydrolysis. The present study is the first where the intrinsic factors involved in the assembly of the DrrAB functional complex have been elucidated. The drrA and drrB genes in the wild type operon have overlapping stop and start codons (ATGA) which indicates translational coupling between the two genes. On insertion of a fortuitous stop codon in DrrA it was shown that the expression of DrrB is coupled to that of the upstream gene drrA. Furthermore, it was observed that a functional complex could be achieved only when the genes were maintained in cis in a translationally coupled manner. Translational regulation in DrrA was found to be involved in the control of optimal levels of DrrB. Inhibitory interactions within drrA sequence were speculated to cause translational arrest at the C terminus of DrrA. A novel assembly domain that forms the interface between DrrA containing the Nucleotide Binding Domain (NBD) and DrrB comprising the TransMembrane Domain (TMD) was found. Based on the data presented in this study a model is proposed for the biogenesis of the DrrAB drug pump. The model suggests that translational coupling between DrrA and DrrB is crucial for functional complex formation. Further, there is evidence of regulation of translation by attenuation in the intergenic region of drrA and drrB. The regulation seems to involve the last 30 nucleotides of the mRNA of drrA and some upstream sequences within drrA that cause translational arrest within the C terminus of DrrA. Since DrrB is translationally coupled to drrA, this translational arrest in conjunction with coupling causes lowering in the levels of DrrB. Finally, since the DrrA-DrrB interaction domain lies in the C terminus of DrrA, only the fully translated DrrA product will be competent to form a complex with DrrB. This interaction between the C terminus of DrrA and the N terminus of DrrB may be crucial for initial targeting of the complex to the membrane. The model is expected to serve as primer and open up an interesting yet insufficiently understood subject of membrane protein biogenesis.

DOI

https://doi.org/10.57709/1063890

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