MuB Protein Research Articles

The mub gene encodes a protein containing three KH domains and is expressed in the mushroom bodies of Drosophila melanogaster.

The ring gland function of Drosophila melanogaster is controlled by the CNS. To identify genes that are active in brain cells and are involved in the ring gland control, we analysed enhancer trap lines with respect to CNS- and/or ring gland-specific lacZ expression in third-instar larvae. From one of the enhancer trap lines, which shows specific lacZ expression in the CNS and prothoracic part of the ring gland, the mub gene was cloned. The gene is strongly expressed in the mushroom bodies throughout development. Nucleotide sequence analysis of cDNA clones revealed a high degree of similarity to vertebrate RNA binding KH domain proteins, suggesting a function of the MUB protein in binding and stabilizing of specific mRNAs in the mushroom bodies. Null mutants of the mub gene do not exhibit a visible mutant phenotype. We speculate, therefore, that the mub gene is involved in learning and memory processes.

Versatile Action of Escherichia coli ClpXP as Protease or Molecular Chaperone for Bacteriophage Mu Transposition

The molecular chaperone ClpX of Escherichia coli plays two distinct functions for bacteriophage Mu DNA replication by transposition. As specificity component of a chaperone-linked protease, it recognizes the Mu immunity repressor for degradation by the peptidase component ClpP, thus derepressing Mu transposition functions. After strand exchange has been promoted by MuA transposase, ClpX alone can alter the conformation of the transpososome (the complex of MuA with Mu ends), and the remodeled MuA promotes transition to replisome assembly. Although ClpXP can degrade MuA, the presence of both ClpP and ClpX in the reconstituted transposition system did not destroy MuA essential for initiation of DNA replication by specific host replication enzymes. Levels of ClpXP needed to overcome inhibition by the repressor did not prevent MuA from promoting strand transfer, and ClpP stimulated alteration of the transpososome by ClpX. Apparently intact MuA was still present in the resulting transpososome, promoting initiation of Mu DNA replication by specific replication enzymes. The results indicate that ClpXP can discriminate repressor and MuA in the transpososome as substrates of the protease or the molecular chaperone alone, degrading repressor while remodeling MuA for its next critical function.

ClpX and MuB interact with overlapping regions of Mu transposase: implications for control of the transposition pathway.

Transposition of phage Mu is catalyzed by an extremely stable transposase-DNA complex. Once recombination is complete, the Escherichia coli ClpX protein, a member of the Clp/Hsp100 chaperone family, initiates disassembly of the complex for phage DNA replication to commence. To understand how the transition between recombination and replication is controlled, we investigated how transposase-DNA complexes are recognized by ClpX. We find that a 10-amino-acid peptide from the carboxy-terminal domain of transposase is required for its recognition by ClpX. This short, positively charged peptide is also sufficient to convert a heterologous protein into a ClpX substrate. The region of transposase that interacts with the transposition activator, MuB protein, is also defined further and found to overlap with that recognized by ClpX. As a consequence, MuB inhibits disassembly of several transposase-DNA complexes that are intermediates in recombination. This ability of MuB to block access to transposase suggests a mechanism for restricting ClpX-mediated remodeling to the proper stage during replicative transposition. We propose that overlap of sequences involved in subunit interactions and those that target a protein for remodeling or destruction may be a useful design for proteins that function in pathways where remodeling or degradation must be regulated.

Bacteriophage Mu: a transposing phage that integrates like retroviruses

Bacteriophage Mu uses a specialized type of genetic recombination called transposition to both integrate its DNA into that of the host and to amplify its DNA during lytic growth. Studies with Mu have defined the basic mechanisms of the DNA cleavage and joining reactions needed for transposition. These mechanisms appear to be used by many transposable elements that populate prokaryotic and eukaryotic hosts. Mu transposition is also very similar to retroviral integration. This conservation in the reaction mechanism is reflected in the transposition proteins. Mu transposase carries an acidic amino acid motif which is required for catalysis that is found in many other transposases and the retroviral integrases. Regulation of Mu transposition by the controlled assembly of the active transposase tetramer and by interactions between the transposase and MuB protein are also discussed.

MuB protein allosterically activates strand transfer by the transposase of phage Mu

The MuA and MuB proteins collaborate to mediate efficient transposition of the phage Mu genome into many DNA target sites. MuA (the transposase) carries out all the DNA cleavage and joining steps. MuB stimulates strand transfer by activating the MuA-donor DNA complex through direct protein-protein contact. The C-terminal domain of MuA is required for this MuA-MuB interaction. Activation of strand transfer occurs irrespective of whether MuB is bound to target DNA. When high levels of MuA generate a pool of free MuB (not bound to DNA) or when chemical modification of MuB impairs its ability to bind DNA, MuB still stimulates strand transfer. However, under these conditions, intramolecular target sites are used exclusively because of their close proximity to the MuA-MuB-donor DNA complex.

Structure-function relationships in the transposition protein B of bacteriophage Mu.

The B-protein of phage Mu, which is required for high frequency intermolecular transposition in vivo, shows ATPase activity in vitro, binds nonspecifically to DNA, and stimulates intermolecular strand transfer. To elucidate the structural bases for B-protein function, it was subjected to limited proteolysis with two different proteases, trypsin and chymotrypsin. The resulting fragments were mapped by amino acid sequencing. These data show that the B-protein is organized in two domains: an amino-terminal domain of 25 kDa and a carboxyl-terminal domain of 8-kDa. A fragment analogous to the amino-terminal domain, produced by deleting the 3' end of a cloned B gene, proved to be insoluble and had to be renatured after elution from a sodium dodecyl sulfate gel. The renatured protein retains ATP-binding activity and to a lesser extent the DNA-binding activity of the MuB protein, but is unable to hydrolyze ATP or function in transposition. We also show in this study that efficient DNA-strand transfer by the B-protein occurs even in the absence of a detectable ATPase activity or in the presence of adenosine 5'-O-(thio)triphosphate (ATP gamma S).