2. \" Mycobacterium tuberculosis PhoP of the PhoP-PhoR two-component signaling s

Question

2. "Mycobacterium tuberculosis PhoP of the PhoP-PhoR two-component signaling system orchestrates a complex transcription program and is essential for the growth and virulence of the tubercle bacillus. PhoP comprises a phosphorylation domain at the amino-terminal half and a DNA-binding domain in the carboxy-terminal half of the protein. We show here that the protein recognizes a 23-bp sequence of the phoP upstream region comprising two adjacent direct repeat motifs believed to promote transcription regulation.
    --Gupta et al. (2009) J Bacteriol. 191(24):7466-7476.
a. The helix-turn-helix motif allows for DNA binding in which groove of DNA?
b. Why does the major groove of DNA allow for more sequence-specific binding than does the minor groove?
c. The authors reported that proteins similar to PhoP form were thought to form head-to-head dimers. The dimerization would prevent it from interacting with the major groove of DNA. If the dimer could only bind the minor groove of DNA, how would that affect the specificity of DNA sequence-specific binding?
d. The authors found that with PhoP, the monomers find bind DNA and then the dimerization occurs. Why would that be necessary for proper sequence-specific recognition?

Explanation / Answer

Ans 1) The helix turn helix motif allows for the DNA binding and bind to the direct tandem repeat sites and inverted repeats of the DNA. It helps in binding to the major groove of the DNA.

Ans 2) The major groove is known for more sequence specific recognition as it binds asymmetrically to the DNA. The monomers basically have their recognition helix in the major groove and this makes the binding more specific towards the major groove as compared to the minor groove. The PhoP tends to bind head to head on the tandem DNA repeats and here one of the repeats is recognized backwards.

Ans 3) The head to head dimerization prevents it from interacting it with the major groove. The dimerization can be stimulated by DNA binding to the major groove and it is more specific to binding to the major groove. DNA is found to be stimulating to the dimerization of the PhoP. In case of PhoP it is non specific and does not bind to the major groove. If it binds to the minor groove, it failed to show a DNA dependent stimulation of the cross linking of the PhoP like proteins and hence dimerization did not take place.

Ans 4) The PhoP needs to bind to specific sequence for the dimerization to take place and it is because without binding to specific sequence, the DNA is not able to stimulate the initiation of dimer formation. If it doesnot bind the specific sequence, dimerization does not take place. The cys-21 residue faces away from each other and makes only minor portion of the protein available for cross linking.

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