Mu transposition occurs within a large protein-DNA complex called a transpososome. This stable complex includes four subunits of MuA transposase, each contacting a 22-base pair recognition site located near an end of the transposon DNA. These MuA recognition sites are critical for assembling the transpososome. Here we report that when concentrations of Mu DNA are limited, the MuA recognition sites permit assembly of transpososomes in which non-Mu DNA substitutes for some of the Mu sequences. These "hybrid" transpososomes are stable to competitor DNA, actively transpose the non-Mu DNA, and produce transposition products that had been previously observed but not explained. The strongest activator of non-Mu transposition is a DNA fragment containing two MuA recognition sites and no cleavage site, but a shorter fragment with just one recognition site is sufficient. Based on our results, we propose that MuA recognition sites drive assembly of functional transpososomes in two complementary ways. Multiple recognition sites help physically position MuA subunits in the transpososome plus each individual site allosterically activates transposase.Transposons are found in all the biological kingdoms, and some perform specialized functions. For example, the machinery that initiates V(D)J recombination likely evolved from a transposon (1, 2), and the cDNA of HIV and other retroviruses integrate into host cell DNA through mechanisms nearly identical to transposition (3). The genome of bacteriophage Mu is a transposon that uses transposition both to integrate into the DNA of a new host cell and to replicate before lysis. Like most DNA rearrangements, transposition is a complex, multi-step process, requiring numerous DNA sequence elements. Studies of bacteriophage Mu have been central to our understanding of both the fundamental mechanisms and the complexities of DNA transposition.Phage Mu encodes a transposase, MuA, that transfers the Mu genome from one DNA location (the transposition donor) to a new location (the transposition target) (4, 5). During transposition, transposase performs two principle reactions: DNA cleavage and DNA strand transfer. During cleavage, the donor DNA is nicked twice, once at each 3Ј end of the Mu genome. During strand transfer, the cleaved transposon ends are inserted into neighboring sites on the two target strands.Little or no specific sequence information is needed on the target DNA (6), but the Mu DNA provides many sequence cues for transposition (Fig. 1). For example, the last two nucleotides at either 3Ј end of the Mu DNA, the cleavage sites, have the sequence 5Ј-CA. Also near each end of the Mu DNA are three recognition sites, distinct from the cleavage sites, which share a 22-base pair consensus sequence. The recognition sites are referred to as R1, R2, and R3 on the right end and L1, L2, and L3 on the left end ( Fig.

Abstract. Apparent physical interaction between peachloroplast (Pisum sativum L.) glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and aldolase (EC 4.1.2.13) is seen in phase-partitioning, fluorescent-anisotropy and isoelectric-focusing experiments. Similarly, results obtained in phase-partitioning and isoelectric-focusing experiments indicate physical interaction between aldolase and triose-phosphate isomerase (EC 5.3.1.1). Kinetic experiments suggest that both aldolase-bound glyceraldehyde-3-phosphate and triose-phosphate isomerase-bound glyceraldehyde-3-phosphate can act as substrate for glyceraldehyde-3-phosphate dehydrogenase. These results are consistent with the notion that there is interaction between these three enzymes both during photosynthetic CO 2 fixation and during glycolysis in the chloroplast.

Transposition of bacteriophage Mu uses two DNA cleavage sites and six transposase recognition sites, with each recognition site divided into two half-sites. The recognition sites can activate transposition of non-Mu DNA sequences if a complete set of Mu sequences is not available. We have analyzed 18 sequences from a non-Mu DNA molecule, selected in a functional assay for the ability to be transposed by MuA transposase. These sequences are remarkably diverse. Nonetheless, when viewed as a group they resemble a Mu DNA end, with a cleavage site and a single recognition site. Analysis of these "pseudo-Mu ends" indicates that most positions in the cleavage and recognition sites contribute sequence-specific information that helps drive transposition, though only the strongest contributors are apparent from mutagenesis data. The sequence analysis also suggests variability in the alignment of recognition half-sites. Transposition assays of specifically designed DNA substrates support the conclusion that the transposition machinery is flexible enough to permit variability in half-site spacing and also perhaps variability in the placement of the recognition site with respect to the cleavage site. This variability causes only local perturbations in the protein-DNA complex, as indicated by experiments in which altered and unaltered DNA substrates are paired.Some of the most fundamental of biological processes occur within nucleoprotein complexes. These processes include recombination, replication, transcription, and RNA splicing. The nucleoprotein complexes are often large and elaborate, containing features that permit sophisticated regulation. As a result, dissecting the chemistry of interactions within a complex is a challenging task, but one that is critical to understanding biological function.Transposition of bacteriophage Mu, like that of other transposons, occurs within complexes called transpososomes. Transpososomes mediate at least two sequential chemical reactions, on a pathway toward transferring the transposon DNA from one site to another. The two reactions are: (i) DNA cleavage, in which a nick is introduced precisely at the end of the transposon, on the 3Ј strand and (ii) DNA strand transfer, in which the nicked strand is joined to a separate DNA molecule called the target (1, 2). The reaction sites on the transposon DNA, or "donor DNA," are defined by specific DNA sequences, but Mu target sites are not very sequence-specific (3).Transpososomes contain multiple subunits of a transposase protein, bound to DNA sequences from both of the transposon's ends (1). These protein-DNA complexes are also called "synaptic complexes" because they bring together the two ends of the transposon DNA. The phage Mu transposase, MuA, is monomeric in solution but forms a tetramer upon binding to specific DNA recognition sites near the transposon ends (4 -6).Each end of the Mu transposon has three MuA recognition sites: R1, R2, and R3 on the right end and L1, L2, and L3 on the left, not all of which are essential for transposition...

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Members of the transposase͞retroviral-integrase superfamily use a single active site to perform at least two reactions during transposition of a DNA transposon or a retroviral cDNA. They hydrolyze a DNA sequence at the end of the mobile DNA and then join this DNA end to a target DNA (a reaction called DNA strand transfer). Critical to understanding the mechanism of recombination is elucidating how these distinct reactions are orchestrated by the same active site. Here we find that DNA substrates terminating in a dideoxynucleotide allow Mu transposase to hydrolyze a target DNA, combining aspects of both natural reactions. Analyses of the sequence preferences for target hydrolysis and of the structure of the cleaved product indicate that this reaction is promoted by the active site in the conformation that normally promotes DNA strand transfer. Dissecting the DNA requirements for target hydrolysis reveals that the ribose of the last nucleotide of the Mu DNA activates transposase's catalytic potential, even when this residue is not a direct chemical participant. These findings provide insight into the molecular mechanism insuring that DNA strand transfer ordinarily occurs rather than inappropriate DNA cleavage. The required presence of the terminal nucleotide in the transposase active site creates a great advantage for the attached 3OH to serve as nucleophile.