Conformational dynamics of a transposition repressor in modulating DNA binding

Sadananda, S.; O'Handley, Diane; Nakai, Hiroshi

doi:10.1006/jmbi.2001.4957

Cited by 11 publications

(19 citation statements)

References 27 publications

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“…Neither the C17A nor the added C-terminal cysteine residue have any effect on DNA binding and protease sensitivity of Rep and Vir (supplemental Fig. S1, A-C), as previously described (9,12). The repressor protein with the C17A mutation and the added C-terminal cysteine (i.e.…”

Section: Methodsmentioning

confidence: 99%

“…MIANS fluorescence was monitored at an excitation wavelength of 330 nm unless otherwise stated with slit widths set to 3 nm. Fluorescence resonance energy transfer from the two tryptophan residues of the Rep DBD (Trp-16 and Trp-44) to MIANS attached to repressor C terminus was measured as previously described (9). Reaction mixtures contained 0.7 M repressor protein with 7.6 M MIANS and 6.2 g/ml pGG215 as indicated.…”

Section: Methodsmentioning

confidence: 99%

“…Attachment of MIANS to the proteins and quantification of bound MIANS were performed as described previously (9). A 3-fold excess of MIANS (4.5 M) was added to each reaction mixture in Buffer A, which was then incubated at 37°C unless otherwise indicated.…”

Section: Methodsmentioning

confidence: 99%

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Activation of a Dormant ClpX Recognition Motif of Bacteriophage Mu Repressor by Inducing High Local Flexibility

Marshall‐Batty

Nakai

2008

Journal of Biological Chemistry

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The C-terminal domain (CTD) of bacteriophage Mu immunity repressor (Rep) regulates DNA binding by the N-terminal domain and degradation by ClpXP protease. Five residues at the Rep C terminus (CTD5) can serve as a ClpX recognition motif, but it is dormant unless activated, a state that can be induced by the presence of dominant-negative mutant repressors (Vir). Conversion of Rep to ClpXP-sensitive form was associated with not only increased exposure of CTD5 to solvent but also increased CTD motion or flexibility as measured by fluorescence anisotropy. CTD mutations (V183S, K193S, and V196S) promoting ClpXP resistance without destroying the recognition motif prevented increased CTD motion induced by Vir. Suppression of ClpXP protease resistance conferred by the V196S mutation also correlated with restoration of CTD motion. The temperature-sensitive R47Q mutation present in cis within the DNA-binding domain restored ClpXP protease sensitivity to the V196S mutant, and anisotropy analysis indicated that R47Q allows the V196S CTD to gain increased flexibility when Vir was present. The results indicate that the CTD functions to turn the recognition motif on and off, most likely by modulating flexibility of the domain that harbors the ClpX recognition motif, suggesting a general mechanism by which proteins can regulate their own degradation.Bacteriophage Mu immunity repressor (Rep) 3 establishes and maintains lysogeny by binding to Mu DNA segments (O1, O2, and O3) that act as both operator (1, 2) and transposition enhancer (3, 4), shutting down transposition functions necessary for Mu replication. Certain physiological conditions, such as starvation or entry into stationary phase (S derepression), can promote degradation or inactivation of the repressor, leading to derepression of these transposition functions (5-8). The C-terminal domain (CTD) of Rep not only contains the recognition motif for initiating proteolysis but also modulates association of the N-terminal DNA-binding domain (DBD) with DNA.In the wild-type repressor (Rep), the CTD is located in close proximity to the DBD (9), a state that we refer to as the closed conformation and that may sterically inhibit interactions of the DBD with DNA. Upon DNA binding, the CTD moves away from the DBD (open conformation). At elevated temperatures, the CTD of temperature-sensitive (ts) repressors, such as the cts62 repressor, which has a R47Q mutation in the DBD, fails to move, and DNA binding is prevented (9). The CTD plays a major role in eliciting the temperature-sensitive DNA binding properties of these mutants; deletions (10) and single amino acid replacements (11) within the CTD can suppress ts mutations to restore stable binding at elevated temperatures. The interconversion of repressor between protease-sensitive and protease-resistant forms also correlates with movement of the CTD; however, the relevant property affected in this interconversion is not the proximity of the CTD to the DBD but rather features such as the exposure of the CTD to solvent (12). Neverthe...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Activation of a Dormant ClpX Recognition Motif of Bacteriophage Mu Repressor by Inducing High Local Flexibility

Marshall‐Batty

Nakai

2008

Journal of Biological Chemistry

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“…As Rep binds to DNA, its CTD moves away from the DBD, whereas the CTD of the temperature-sensitive DBD mutant cts62Rep fails to do so at the elevated temperature (17). The close proximity of the CTD to the DBD is associated with low DNA binding affinity, suggesting that the CTD may sterically inhibit interactions of the DBD with DNA.…”

mentioning

confidence: 99%

“…We suggest that the ClpX recognition motif of Rep may be activated when attached to disordered or flexible domains that confer ligand flexibility. (27), ClpP (28), Rep, and Vir3060 proteins (17,18) were purified as described previously. Green fluorescent protein (GFP) fusion proteins, which contain an N-terminal His 10 tag, were overproduced in strain BL21(DE3) (Novagen) from plasmid vectors described below.…”

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A Context-dependent ClpX Recognition Determinant Located at the C Terminus of Phage Mu Repressor

Defenbaugh

Nakai

2003

Journal of Biological Chemistry

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The bacteriophage Mu immunity repressor is a conformationally sensitive sensor that can be interconverted between forms resistant to and sensitive to degradation by ClpXP protease. Protease-sensitive repressor molecules with an altered C-terminal sequence promote rapid degradation of the wild-type repressor by inducing its C-terminal end to become exposed. Here we determined that the last 5 C-terminal residues (CTD5) of the wild-type repressor contain the motif required for recognition by the ClpX molecular chaperone, a motif that is strongly dependent upon the context in which it is presented. Although attachment of the 11-residue ssrA degradation tag to the C terminus of green fluorescent protein (GFP) promoted its rapid degradation by ClpXP, attachment of 5-27 C-terminal residues of the repressor failed to promote degradation. Disordered peptides derived from 41 and 35 C-terminal residues of CcdA (CcdA41) and thioredoxin (TrxA35), respectively, activated CTD5 when placed as linkers between GFP and repressor C-terminal sequences. However, when the entire thioredoxin sequence was included as a linker to promote an ordered configuration of the TrxA35 peptide, the resulting substrate was not degraded. In addition, a hybrid tag, in which CTD5 replaced the 3-residue recognition motif of the ssrA tag, was inactive when attached directly to GFP but active when attached through the CcdA41 peptide. Thus, CTD5 is sufficient to act as a recognition motif but has requirements for its presentation not shared by the ssrA tag. We suggest that activation of CTD5 may require presentation on a disordered or flexible domain that confers ligand flexibility.

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Protein Fluorescence

2006

Principles of Fluorescence Spectroscopy

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Conformational dynamics of a transposition repressor in modulating DNA binding

Cited by 11 publications

References 27 publications

Activation of a Dormant ClpX Recognition Motif of Bacteriophage Mu Repressor by Inducing High Local Flexibility

Activation of a Dormant ClpX Recognition Motif of Bacteriophage Mu Repressor by Inducing High Local Flexibility

A Context-dependent ClpX Recognition Determinant Located at the C Terminus of Phage Mu Repressor

Protein Fluorescence

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