The T4 Phage DNA Mimic Protein Arn Inhibits the DNA-binding Activity of the Bacterial Histone-like Protein H-NS
Date Issued
2015
Date
2015
Author(s)
Ho, Chun-Han
Abstract
Organisms using DNA as their hereditary substance, the genetic information of these DNA sequence can be expressed and regulated by DNA-related functional proteins, such as transcriptional factors and DNA-binding proteins. Around a decade ago, a new category of control factors of DNA-related functional proteins called the DNA mimic protein had been identified. The DNA mimic protein can inhibit and/or regulate the function and activity of DNA-related functional proteins by its DNA-like shape and unique surface negative charge pattern. Thus, DNA mimic proteins are involved in certain important cellular processes and may be involved in many undiscovered regulation mechanisms. To identify the DNA mimic protein in T4 phage, we focused on the proteins which have the properties found in known DNA mimic proteins: small protein size and low isoelectric point (pI). After the sequence analysis, we found that anti restriction nuclease (Arn) protein may be a DNA mimic protein. By using structural approaches, the crystal structure of Arn was determined. Interestingly, the negative charge distribution of Arn dimer surface is similar to the phosphate group distribution on DNA, implying Arn dimer could act as a DNA mimic. Furthermore, the size and shape of Arn dimer is similar to the DNA mimic protein overcome classical restriction (Ocr). To identify the interaction partner of T4 phage Arn in Escherichia coli (E. coli), the His-pull-down was used and we further discovered that bacterial histone-like nucleoid structuring (H-NS) protein can interact with Arn specifically. Arn/H-NS interaction reveals a novel regulation mechanism. When infecting the E. coli, T4 phage encounters attacks from host’s defense systems. For example, bacteria express some small DNA-binding proteins to bind and entangle foreign or phage DNAs, inducing the gene silencing effect. H-NS is a member of these kind of protein. Thus, in order to infect and replicate in E. coli successfully, T4 phage has to evolve some strategies to overcome the gene silencing effect from H-NS. Therefore, using a DNA mimic protein to inhibit H-NS is a straightforward way for T4 phage. To confirm our hypothesis in this undiscovered function of Arn, electrophoresis mobility shift assay (EMSA) was used and demonstrated that Arn competes with bacteria as well as phage DNA fragments for binding to H-NS. Computer modeling analysis revealed that Arn dimer competes with DNA to interact with the H-NS DNA binding domain via its negatively-charged side. Additionally, in vitro gene expression and electron microscopy analyses further indicated that Arn antagonizes the gene-silencing effect of H-NS on the reporter gene. In summary, we discovered a novel mechanism for phage-bacteria battle from T4 phage, which employs the DNA mimic protein Arn to counteract the H-NS gene-silencing effect by its DNA-like surface.
Subjects
molecular mimicry
DNA mimic protein
anti-bacterial defense system
anti-gene silencing
nucleoid
Type
thesis
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