Modulation of phage Mu repressor DNA binding and degradation by distinct determinants in its C‐terminal domain

Abstract

SummaryRapid degradation of the bacteriophage Mu immunity repressor can be induced in trans by mutant, protease-hypersensitive repressors (Vir) with an altered C-terminal domain (CTD). Genetic and biochemical analysis established that distinct yet overlapping determinants in the wild-type repressor CTD modulate Vir-induced degradation by Escherichia coli ClpXP protease and DNA binding by the N-terminal DNA-binding domain (DBD). Although deletions of the repressor C-terminus resulted in both resistance to ClpXP protease and suppression of a temperaturesensitive DBD mutation ( cts62 ), some cysteinereplacement mutations in the CTD elicited only one of the two phenotypes. Some CTD mutations prevented degradation induced by Vir and resulted in the loss of intrinsic ClpXP protease sensitivity, characteristic of wild-type repressor, and at least two mutant repressors protected Vir from proteolysis. One proteaseresistant mutant became susceptible to Vir-induced degradation when it also contained the cts62 mutation, which weakens DNA binding but apparently facilitates conversion to a protease-sensitive conformation. Conversely, this CTD mutation was able to suppress temperature sensitivity of DNA binding by the cts62 repressor. The results suggest that determinants in the CTD not only provide a cryptic ClpX recognition motif but also direct CTD movement that exposes the motif and modulates DNA binding.

Transcriptional Regulation of Cannabinoid Receptor-1 Expression in the Liver by Retinoic Acid Acting via Retinoic Acid Receptor-γ

“…Autoradiograms of Western blots were scanned and quantified using Quantity One software (Bio-Rad). All blots were normalized to the loading control ␤-actin (21).…”

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

“…Bacterial Strains, Plasmids, and Proteins-Expression vectors pHS502 (Rep), pHS504 (Vir3060), pGTN206 (VirC17A, where the single cysteine at residue 17 has been replaced with an alanine), pGTN204 (RepC197, which has an engineered C-terminal cysteine at residue 197 and the C17A alteration), and pGTN211 (VirC192; engineered C-terminal cysteine at residue 192 and the C17A alteration) were used to express the indicated proteins, which were purified as previously described (11)(12)(13). The QuikChange TM site-directed mutagenesis kit (Stratagene) was used to introduce single amino acid replacements in the repressor coding sequence.…”

“…Reaction mixtures (30 l) contained 20 g/ml pGG215 with varying repressor concentrations (0 -8 M) in 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM magnesium chloride, 1 mM dithiothreitol and were incubated at 30°C and 42°C for 15 min prior to HindIII digestion and agarose gel electrophoresis (11). All DNA binding assays were performed at least three times, and representative results are shown.…”

“…In addition, cysteine scan mutagenesis has indicated that mutations at 8 individual positions in the CTD (Ile-170 to Val-196 or CTD27; Fig. 1A) confer ClpXP resistance in the presence of Vir (11). Six of these mutations are located outside the CTD5 recognition motif, implying that the CTD may provide more than just a recognition motif.…”

“…Six of these mutations are located outside the CTD5 recognition motif, implying that the CTD may provide more than just a recognition motif. Two of the CTD mutations (V183C and V196C) promoted ClpXP resistance even after heat denaturation (11), raising the question of whether exposure of CTD5 is sufficient to promote degradation of Rep. Furthermore, when CTD5 is attached to the C terminus of green fluorescent protein (GFP), its ability to promote GFP degradation is highly dependent upon the presence of a disordered linker (e.g.…”