Oligomeric structure of the repressor of the bacteriophage Mu early operon

Abstract

The regulation of the lytic and lysogenic development in the life cycle of bacteriophage Mu is regulated in part by its repressor, c, which binds to three operator sites, O1, O2 and O3, overlapping two divergent promoters. The oligomeric structure of this repressor protein was investigated by hydrodynamic and biochemical methods. Size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering, crosslinking and direct electron microscopy observations suggest that c exists primarily as a hexamer with a molecular mass of 120Ϫ140 kDa at low concentrations, i.e. in the 10-µM range. This molecule undergoes a self-assembly process leading to dodecamers and higher order species as the concentration is further increased in a manner depending on the nature of the solvent. Our results also suggest that these species have an elongated structure, and a possible arrangement of the subunits within the hexamer is proposed. The implication of this unusual quaternary structure for a repressor in its interaction with the operator sites O1 and O2 remains to be elucidated.Keywords : Mu repressor; protein quaternary structure ; oligomerisation.In addition to its role in the establishment and maintenance The assembly of higher order nucleoprotein structures is essential for the control of many key cellular processes including of the lysogenic state, the region covering O1 and O2 acts as an enhancer (internal activating sequence, IAS) during the assemreplication, recombination, repair, transposition and transcription. Such complex structures are established in regulating the bly of the first stable intermediate in transposition, the type-0 complex (stable synaptic complex, SSC; [10Ϫ13]). This process expression of the early operon of bacteriophage Mu which controls the choice between the lytic and lysogenic pathways in involves the transient binding of transposase, pA, to the IAS through its N-terminal region (76 amino acids; [14]) in addition Escherichia coli. This process is regulated by the phage repressor, c, which binds to a 200-bp region located 1 kb from the left to binding to the left (attL) and right (attR) ends of the phage genome. The N-terminal region of pA shares about 44% simiend of the phage genome and composed of three operator sites, O1, O2 and O3, overlapping two divergent promoters, Pe and larity with that of the repressor [15, 16], and it has been demonstrated that the repressor also inhibits the transposition process Pc (Fig. 1). Transcription from Pe, located in O2, leads to the expression of early lytic functions including ner, the transposase by competing with the transposase N-terminal domain for its binding to IAS, thus impairing synapsis leading to the first pA, and an accessory protein pB which stimulates the transposition process. Transcription from Pc, which is located in O3, transposition intermediate [17, 14].In an attempt to determine the organisation of the complex leads to the synthesis of the repressor c. The operator sites O1, O2 and O3 contain 4, 3 and 2 imperfectly repeated...

trans-Targeting of the Phage Mu Repressor Is Promoted by Conformational Changes That Expose Its ClpX Recognition Determinant

“…32 PRepNK and an unlabeled repressor protein were first mixed and allowed to incubate for 10 mins at 37°C before addition of 1 g trypsin. Digestion was allowed to proceed at the same temperature for 1 h, and then reactions were stopped by the addition of phenylmethylsulfonyl fluoride to 2% (w/v) final concentration.…”

“…trans-Targeting proteins such as Vir may function to expose such a determinant in Rep to promote its degradation. The self-associating property of repressor molecules, which exists in solution as a hexamer or higher order oligomer (32,33), may promote Rep-Vir interactions for this process.…”

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

“…Even after considering other possible values for (p) and [] (37), our calculations indicated that cWHOLE only varied from 8.6 to 10.3 ns. More importantly, correlation times are longer for larger molecules or complexes, which would have more restricted motion, and Mu repressor is known to exist in hexameric and even higher order oligomeric forms in solution (41,42). Such oligomeric complexes would make up a complex of at least 130 kDa; therefore, the cWHOLE of the repressor protein is more likely 54 ns or more.…”

“…cWHOLE would be longer for larger molecules or complexes, which would rotate more slowly. Since the repressor is known to assume a hexameric or higher order oligomeric structure in solution (41,42), the rotational correlation time is most likely 54 ns or greater. However, FIGURE 5.…”

Trans‐targeting of protease substrates by conformationally activating a regulable ClpX‐recognition motif

“…The product of the c gene of bacteriophage Mu encodes a 196‐residue transposition repressor (Krause and Higgins, 1986; Leung et al ., 1989; Mizuuchi and Mizuuchi, 1989) that is a ClpX substrate (Welty et al ., 1997). It exists in solution as a hexamer or higher order oligomers (Rousseau et al ., 1996; Alazard et al ., 1998), a property that may be important not only for its cooperative binding to Mu operator DNA (Vogel et al ., 1991) but also for communication of conformational states between repressor isoforms (Marshall‐Batty and Nakai, 2003). Mu repressor regulates the decision between lysogeny and lytic development; degradation or inactivation of the Mu repressor leads to expression of transposition functions MuA and MuB, promoting Mu DNA synthesis through replicative transposition.…”