Bacterial one-hybrid system

In parallel, a library of randomized oligonucleotides representing potential TF target sequences are cloned into a separate vector containing the selectable genes HIS3 and URA3.

If the DNA-binding domain (bait) binds a potential DNA target site (prey) in vivo, it will recruit RNA polymerase to the promoter and activate transcription of the reporter genes in that clone.

The other plasmid contains a region of randomized sequence representing potential binding sites (prey) which, if bound to by the chimeric fusion product, drives expression of downstream reporter genes.

This reporter region facilitates both positive and negative selection by HIS3 and URA3, respectively, which together allow for isolation of the prey containing the true DNA target sequence.

Designing the fusion construct (bait) to the omega, rather than the alpha, subunit of RNA polymerase has recently been favoured in order to improve the chimera’s stereochemistry and dynamic range.

[6] A zinc-finger domain on the fusion construct and its corresponding DNA target site, adjacent to the randomized prey sequence, has also been added to the increases affinity and specificity of protein–DNA interactions.

SELEX, another system commonly used to identify the target nucleic acids for DNA-binding proteins, requires multiple rounds of selection.

In contrast, the bacterial one-hybrid system requires just one round of in vitro selection and also offers a low-tech alternative to microarray-based technologies.

Second, some eukaryotic factors may not express or fold efficiently in the bacterial system, attributed to differing regulatory networks and transcriptional machinery.

[1][3] B1H system provides a tool in our arsenal for identifying the DNA-binding specificities of transcription factors and thus predicting their target genes and genomic DNA regulatory elements.

It also allows for examination of the effects of protein–protein interactions on DNA binding, which may further guide the prediction of cis regulatory modules based on binding-site clustering.

[8] Implications for medical research are evident from another study that used the B1H system to identify the DNA-binding specificity of a transcriptional regulator for a gene in Mycobacterium tuberculosis.

Figure 1: Overview of the bacterial one-hybrid system . A library of randomized nucleotides representing potential transcription factor binding sites (10–20 base pairs), the “prey”, is cloned upstream of HIS3 and URA3, positive and negative selectable markers, respectively. If the transcription factor binds to the randomized region it will recruit RNA polymerase via direct fusion to the α- or ω-subunit and thus activate transcription of the downstream reporter genes. The host E. coli strain being used must lack the bacterial homologues of these biosynthetic genes (HIS3 and URA3 ).
Figure 2: The bacteria one-hybrid system requires two customized plasmids: (a) the “bait” vector (pB1H1 or pB1H2) which expresses the DNA-binding domain as a fusion construct with a subunit (α or ω) of RNA polymerase, and (b) a reporter plasmid (pH3U3) containing selectable markers and a binding-site library, or “prey,” which will potentially interact with the "bait.”
Figure 3: Overview of the B1H negative and the positive selection procedure. The DNA segment labeled 'RR' refers to the randomized region on prey vectors. Self-activating sequences are purged from the original binding-site library by attempting to grow prey-transformed E.coli on medium containing 5-FOA, a compound that is cleaved into a toxin by URA3. The resulting purified prey library is then transformed with the bait plasmid and grown on minimal medium lacking histidine in order to positively select for actively expressed reporter vectors. This medium is supplemented with varying concentrations of 3-AT, a competitive inhibitor of HIS3, to elucidate the binding affinities of the transcription factor to each particular target sequence. The randomized regions from surviving colonies are then isolated and sequenced prior to motif-finding analysis (ex, MEME, BioProspector). Finally, optimal and tolerated bases at the key binding-site positions are generated