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An Unusual Phage Repressor Encoded by Mycobacteriophage BPs.

Villanueva VM, Oldfield LM, Hatfull GF - PLoS ONE (2015)

Bottom Line: We show here that both forms of the repressor bind similarly to the 33-34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution.BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site.The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, 15260, United States of America.

ABSTRACT
Temperate bacteriophages express transcription repressors that maintain lysogeny by down-regulating lytic promoters and confer superinfection immunity. Repressor regulation is critical to the outcome of infection-lysogenic or lytic growth-as well as prophage induction into lytic replication. Mycobacteriophage BPs and its relatives use an unusual integration-dependent immunity system in which the phage attachment site (attP) is located within the repressor gene (33) such that site-specific integration leads to synthesis of a prophage-encoded product (gp33103) that is 33 residues shorter at its C-terminus than the virally-encoded protein (gp33136). However, the shorter form of the repressor (gp33103) is stable and active in repression of the early lytic promoter PR, whereas the longer virally-encoded form (gp33136) is inactive due to targeted degradation via a C-terminal ssrA-like tag. We show here that both forms of the repressor bind similarly to the 33-34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution. The BPs gp33103 repressor binds to five regulatory regions spanning the BPs genome, and regulates four promoters including the early lytic promoter, PR. BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site. The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome.

No MeSH data available.


Related in: MedlinePlus

BPs gp33103 binds with variable affinities to a series of end-deletion substrates.Substrates were PCR amplified from the BPs 33–34 intergenic region using primers located at regular intervals from each end to sequentially shorten the 33–34 intergenic region from each end (S1 Table). The sequences included in each substrate are shown as horizontal black lines either above or below a schematic representation of the 33–34 intergenic region. Genes 33 and 34 are represented by blue and orange arrows respectively, and OR and ORep are shown in yellow. Protein concentrations are same as shown in Fig 2A. Protein affinities are shown in Table 1.
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pone.0137187.g004: BPs gp33103 binds with variable affinities to a series of end-deletion substrates.Substrates were PCR amplified from the BPs 33–34 intergenic region using primers located at regular intervals from each end to sequentially shorten the 33–34 intergenic region from each end (S1 Table). The sequences included in each substrate are shown as horizontal black lines either above or below a schematic representation of the 33–34 intergenic region. Genes 33 and 34 are represented by blue and orange arrows respectively, and OR and ORep are shown in yellow. Protein concentrations are same as shown in Fig 2A. Protein affinities are shown in Table 1.

Mentions: To further examine the parts of the 33–34 intergenic region required for binding we generated a series of substrates containing progressive deletions from each end and tested them for binding of gp33103 (Fig 4). One notable observation is that the binding patterns with the Mt12 and Mt13 substrates (Figs 1C and 4) are similar, even though OR-R is present in Mt13 but absent from Mt12. The affinity for Mt12 is slightly reduced relative to that for Mt13, but we assume that the slowest migrating complexes (C4) have similar stoichiometries, with each containing a dimer or tetramer of gp33103 bound at OR in addition to binding elsewhere in the substrate. One of the Mt13 complexes (C3) appears to be absent from the Mt12 substrate, and presumably requires OR-R (Figs 1 and 4). Complete removal of both OR-L and OR-R (substrates Mt10 and Mt11) does not completely eliminate binding and complexes are observed albeit with reduced binding affinity (Fig 4; Table 1), reflecting weaker binding to the left part of the substrate.


An Unusual Phage Repressor Encoded by Mycobacteriophage BPs.

Villanueva VM, Oldfield LM, Hatfull GF - PLoS ONE (2015)

BPs gp33103 binds with variable affinities to a series of end-deletion substrates.Substrates were PCR amplified from the BPs 33–34 intergenic region using primers located at regular intervals from each end to sequentially shorten the 33–34 intergenic region from each end (S1 Table). The sequences included in each substrate are shown as horizontal black lines either above or below a schematic representation of the 33–34 intergenic region. Genes 33 and 34 are represented by blue and orange arrows respectively, and OR and ORep are shown in yellow. Protein concentrations are same as shown in Fig 2A. Protein affinities are shown in Table 1.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4557955&req=5

pone.0137187.g004: BPs gp33103 binds with variable affinities to a series of end-deletion substrates.Substrates were PCR amplified from the BPs 33–34 intergenic region using primers located at regular intervals from each end to sequentially shorten the 33–34 intergenic region from each end (S1 Table). The sequences included in each substrate are shown as horizontal black lines either above or below a schematic representation of the 33–34 intergenic region. Genes 33 and 34 are represented by blue and orange arrows respectively, and OR and ORep are shown in yellow. Protein concentrations are same as shown in Fig 2A. Protein affinities are shown in Table 1.
Mentions: To further examine the parts of the 33–34 intergenic region required for binding we generated a series of substrates containing progressive deletions from each end and tested them for binding of gp33103 (Fig 4). One notable observation is that the binding patterns with the Mt12 and Mt13 substrates (Figs 1C and 4) are similar, even though OR-R is present in Mt13 but absent from Mt12. The affinity for Mt12 is slightly reduced relative to that for Mt13, but we assume that the slowest migrating complexes (C4) have similar stoichiometries, with each containing a dimer or tetramer of gp33103 bound at OR in addition to binding elsewhere in the substrate. One of the Mt13 complexes (C3) appears to be absent from the Mt12 substrate, and presumably requires OR-R (Figs 1 and 4). Complete removal of both OR-L and OR-R (substrates Mt10 and Mt11) does not completely eliminate binding and complexes are observed albeit with reduced binding affinity (Fig 4; Table 1), reflecting weaker binding to the left part of the substrate.

Bottom Line: We show here that both forms of the repressor bind similarly to the 33-34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution.BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site.The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, 15260, United States of America.

ABSTRACT
Temperate bacteriophages express transcription repressors that maintain lysogeny by down-regulating lytic promoters and confer superinfection immunity. Repressor regulation is critical to the outcome of infection-lysogenic or lytic growth-as well as prophage induction into lytic replication. Mycobacteriophage BPs and its relatives use an unusual integration-dependent immunity system in which the phage attachment site (attP) is located within the repressor gene (33) such that site-specific integration leads to synthesis of a prophage-encoded product (gp33103) that is 33 residues shorter at its C-terminus than the virally-encoded protein (gp33136). However, the shorter form of the repressor (gp33103) is stable and active in repression of the early lytic promoter PR, whereas the longer virally-encoded form (gp33136) is inactive due to targeted degradation via a C-terminal ssrA-like tag. We show here that both forms of the repressor bind similarly to the 33-34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution. The BPs gp33103 repressor binds to five regulatory regions spanning the BPs genome, and regulates four promoters including the early lytic promoter, PR. BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site. The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome.

No MeSH data available.


Related in: MedlinePlus