Team:SDU-Denmark/DialogLink1
Alternative versions of the bacterial two-hybrid system
Another form of two hybrid screening is the bacterial two hybrid system made in E.coli. The system can be used to examine both DNA-protein and protein-protein interactions. Much like the yeast two hybrid system, the bacterial two hybrid system revolves around the interaction between two proteins, which lead to the activation of a reporter-gene like the lacZ-reporter gene. One protein consist of a DNA binding domain (DBD) fused to another domain called interaction domain 1 (ID1). The second protein consists of a part of the E.coli RNA polymerase fused to a domain, interaction domain 2 (ID2). For the activation of lacZ expression to occur there must be an interaction between ID1 and ID2 and the DBD must bind to a DNA binding site (DBS) near the lacZ-gene promoter. The interaction between ID1 and ID2 recruit the RNA polymerase to the lacZ-promoter initiating transcription.
Reference:
SIMON L. DOVE, J. KEITH JOUNG* & ANN HOCHSCHILD. (1997) Activation of prokaryotic transcription through arbitrary protein–protein contacts. Nature 386, 627-630 (10 April 1997).
[Nature]
J. Keith Joung, Elizabeth I. Ramm, and Carl O. Pabo (2000) A bacterial two-hybrid selection system for studying protein–DNA and protein–protein interactions. PNAS. US. vol. 97, (20 june 2000).
[PubMed]
A modified version of the bacterial two hybrid system was presented by Joung and co. This modified two hybrid system consists of does not initiate the transcription of lacZ, but instead that of the HIS3- and aadA-genes, which codes for an enzyme required for the biosynthesis of histidine and the resistance for the antibiotic spectinomyocine. A Zif268 Binding site (ZBP) is positioned upstream from the HIS3-aadA-promoter. The protein binding to the ZBD is Gal11P fused with the 3 zing finger domains from the murine Zif268 protein (ZF1, ZF2, ZF3). Gal11P-Zif268 is replacing the fusion protein DBD-ID1. Bound to the RNA polymerase of E.coli is αGal4-protein. The αGal4-Gal11P-Zif268 interaction lead to the recruitment of the RNA polymerase, which lead to transcription of the HIS3-aadA-genes. This cassette is placed in an E.coli with a deletion in the homologous hisB gene and grown on HIS selective medium. So if there is an interaction between the zinc finger domains and the DNA the HIS3 gene would be transcribed and the cells would grow. This system was then used to examine interactions between randomized zinc finger domains and DNA. One of the 3 zinc finger domains (ZFD) of the Gal11P-Zif268 complex was replaced by a randomized ZFD. If only 2 ZFDs could bind the DNA transcription of the HIS3 gene will be low inhibiting growth, while if all 3 ZFDs bind the DNA the transcription of the gene would be high and the cells would grow. Reference: J. Keith Joung, Elizabeth I. Ramm, and Carl O. Pabo (2000) A bacterial two-hybrid selection system for studying protein–DNA and protein–protein interactions. PNAS. US. vol. 97, (20 june 2000). [PubMed] In our case the system described by Joung is a bit too advanced, but the selection system using E.coli with deficient histidine production could be a viable option in the screening process for our aptamer.