Molecular roadblocks for RpoN binding sites
View Patent ↗The present invention relates to, inter alia, a method for repressing transcription and/or gene expression from RpoN binding sites (or promoters) or cryptic promoters upstream of RpoN binding sites. The method comprises providing an agent that specifically and selectively binds to RpoN promoter sequences to inhibit or repress the expression of genes downstream of that promoter; and contacting the RpoN promoter with the agent. Agents for repressing transcription and/or gene expression from RpoN promoters are also provided. The agent can be a composition that binds specifically to the −24, −12, or −24/−12 site(s) for RpoN promoter interference. Synthetic peptides, vectors, and host cells are also provided.
1. A method for repressing transcription or gene expression from a binding site of interest, wherein the binding site of interest is an RpoN binding site or a cryptic promoter upstream or downstream of an RpoN binding site, comprising the steps of:
(a) providing an agent wherein the agent:
(i) specifically and selectively binds to a sequence comprised in the binding site of interest,
(ii) inhibits or represses expression of genes regulated by the binding site of interest, and
(iii) comprises a heterologous protein comprising a sequence selected from the group consisting of SEQ ID NOS. 10000-10093; and
(b) contacting the agent to the binding site of interest.
2. The method of claim 1 , wherein said contacting comprises contacting the agent to a bacterial cell comprising the binding site of interest, wherein the agent is heterologous to the bacterial cell.
3. The method of claim 1 , wherein the agent binds specifically to a −24, −12, or −24/−12 site for RpoN promoter interference.
4. The method of claim 1 , wherein the RpoN binding site comprises a sequence selected from the group consisting of SEQ ID NOS. 1-9999 and the 3′ to 5′ complementary base pairs thereof.
5. The method of claim 1 , wherein the agent comprises a σ 54 (“sig54”) protein.
6. The method of claim 5 , wherein the agent is provided by heterologously expressing the σ 54 (“sig54”) protein.
7. The method of claim 1 , wherein the agent comprises an RpoN protein comprising a modification to the heptad repeat overlapping the glutamine-rich or acid-rich motifs.
8. The method of claim 1 , wherein the agent binds specifically and selectively to the cryptic promoter.
9. The method of claim 1 , wherein the agent is a synthetic peptide from 20 to 600 amino acids in length.
10. The method of claim 9 , wherein the synthetic peptide comprises at least 20 carboxy terminal amino acids of an RpoN protein.
11. The method of claim 9 , wherein the synthetic peptide comprises the conserved amino acids from an RpoN box of Region III of an RpoN protein through the carboxy terminal amino acid residue of the RpoN protein.
12. The method of claim 9 , wherein the synthetic peptide has 70-75%, 75-80%, 80-85%, 85-90%, 90-95% or 95-100% homology with an RpoN box of Region III of an RpoN protein.
13. The method of claim 11 , wherein Region III comprises a modification of the heptad repeat overlapping the glutamine-rich or acid-rich motifs.