William S. Reznikoff

Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes.

DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene–operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref. 1). However, all in vivo systems have a number of technical limitations. For instance, the transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro transposition systems (for Tn5, Tn7, Mu, Himar1, and Ty1) have been described, which bypass many limitations of in vivo systems. For this purpose, we have developed a technique for transposition that involves the formation in vitro of released Tn5 transposition complexes (TransposomesTM) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency transposition in all tested bacterial species (Escherichia coli, Salmonella typhimurium, and Proteus vulgaris) We also isolated transposition events in the yeast Saccharomyces cerevisiae.

Mobile DNA in obligate intracellular bacteria

The small genomes of obligate intracellular bacteria are often presumed to be impervious to mobile DNA and the fluid genetic processes that drive diversification in free-living bacteria. Categorized by reductive evolution and streamlining, the genomes of some obligate intracellular bacteria manifest striking degrees of stability and gene synteny. However, recent findings from complete genome sequences of obligate intracellular species and their mobile genetic associates favour the abandonment of these wholesale terms for a more complex and tantalizing picture.

Hairpin Formation in Tn5 Transposition

The initial chemical steps in Tn5transposition result in blunt end cleavage of the transposon from the donor DNA. We demonstrate that this cleavage occurs via a hairpin intermediate. The first step is a 3′ hydrolytic nick by transposase. The free 3′OH then attacks the phosphodiester bond on the opposite strand, forming a hairpin at the transposon end. In addition to forming precise hairpins, Tn5 transposase can form imprecise hairpins. This is the first example of imprecise hairpin formation on transposon end DNA. To undergo strand transfer, the hairpin must to be resolved by a transposase-catalyzed hydrolytic cleavage. We show that both precise and imprecise hairpins are opened by transposase. A transposition mechanism utilizing a hairpin intermediate allows a single transposase active site to cleave both 3′ and 5′ strands without massive protein/DNA rearrangements.

The inverted repeats of Tn5 are functionally different.

The inverted repeats of Tn5, which have identical restriction endonuclease cleavage patterns, have different functional properties. They differ with respect to RNA polymerase binding, full promotion of neomycin resistance, the polypeptides coded for by the repeats and their function in the transposition process. There is a week RNA polymerase binding site present in one repeat and not in the other which seems to be important for neomycin resistance. The two inverted repeats code for polypeptides of different molecular weights, with each repeat appearing to encode two polypeptides. The polypeptides from only one of the repeats of Tn5 appear to be absolutely required for Tn5 transposition.

Control of Tn5 Transposition in Escherichia coli Is Mediated by Protein from the Right Repeat

The right repeat in Tn5, which encodes protein absolutely required for transposition, is also capable of inhibiting Tn5 transposition. Analysis of Tn5 mutants indicates that the left repeat is defective in supplying the transposition-inhibition function because of the sequence difference between the repeats located at nucleotide 1443; that the transposition-inhibition activity is a function of the quantity of right-repeat protein synthesis; that the smaller of the right-repeat proteins, protein 2, is sufficient for supplying the transposition-inhibition function (but not for the transposase activity); and that the transposition-inhibition function can act in trans, as opposed to the transposase activity, which functions efficiently only in cis. Gene fusion experiments indicate that the transposition-inhibition activity cannot be explained by autogenous regulation of right-repeat protein synthesis. Finally, immunoprecipitation assays of right-repeat protein-lacZ fusion proteins indicate that protein 2 is synthesized in significantly greater amounts than protein 1 in whole cells. This synthetic ratio may ber important with respect to the control of Tn5 transposition.

Tn5 as a model for understanding DNA transposition.

Tn5 is an excellent model system for understanding the molecular basis of DNA‐mediated transposition. Mechanistic information has come from genetic and biochemical investigations of the transposase and its interactions with the recognition DNA sequences at the ends of the transposon. More recently, molecular structure analyses of catalytically active transposase; transposon DNA complexes have provided us with unprecedented insights into this transposition system. Transposase initiates transposition by forming a dimeric transposase, transposon DNA complex. In the context of this complex, the transposase then catalyses four phosphoryl transfer reactions (DNA nicking, DNA hairpin formation, hairpin resolution and strand transfer into target DNA) resulting in the integration of the transposon into its new DNA site. The studies that elucidated these steps also provided important insights into the integration of retroviral genomes into host DNA and the immune system V(D)J joining process. This review will describe the structures and steps involved in Tn5 transposition and point out a biologically important although surprising characteristic of the wild‐type Tn5 transposase. Transposase is a very inactive protein. An inactive transposase protein ensures the survival of the host and thus the survival of Tn5.

The functional differences in the inverted repeats of Tn5 are caused by a single base pair nonhomology

The inverted repeats of Tn5 are functionally different. One repeat codes for larger polypeptides, which are required for transposition. The other repeat has a better promoter for the neomycin resistance gene in the region of the repeat near the unique sequences. These dissimilarities are now shown to be caused by a single base pair difference. This change both creates a better promoter sequence and codes for part of a new UAA nonsense codon. Mutants in which the DNA sequence of a repeat is altered only at this base pair are shown to function like the opposite repeat. Furthermore, it is possible to suppress the UAA nonsense codon with an ochre suppressor, making the previously abbreviated polypeptides functional in transposition.

The lactose operon‐controlling elements: a complex paradigm

The lactose‐controlling elements have been considered to be the simple paradigm of a cis‐acting genetic regulatory system, containing a promoter whose activity is modulated by an operator and a catabolite gene activator protein (CAP)‐binding site. The reality is considerably more complex. We now know that transcription is negatively regulated as a result of the repressor binding to three binding sites: the operator, a secondary repressor‐binding site within the lacZ gene and a tertiary repressor‐binding site upstream near lacl. In addition to the promoter, the lac‐controlling elements contain five promoter‐like elements. The physiological role, if any, of these promoter‐like elements is not clear, although three of them can be activated by single base pair changes to give high levels of in vivo expression. Finally, the positive activator protein CAP has been found to bind to a secondary site which is coincident with the operator. No role has been identified for this secondary CAP‐DNA complex.

Effect of dam methylation on Tn5 transposition

The effect of dam methylation on Tn5 transposition was investigated by analyses of mutations in the host (Escherichia coli) and the element. Wild-type elements transposed at a higher frequency and showed higher levels of transposase expression in a dam-host. Mutations were made in the promoter region of the transcript that codes for the transposase. Transposition and transposase levels from these mutants were independent of the host methylation system. Measurements of the amount of RNA support the hypothesis that dam methylation exerts its effect on Tn5 transposition by modulating the frequency of transcriptional initiation of the transposase gene. Since Tn5 transposition increases when the transposase levels increase, at normal concentrations the amount of transposase is a rate-limiting factor that determines the transposition frequency of Tn5. Transposition of IS50, one of the insertion sequences that constitutes Tn5, is also sensitive to dam methylation by a second mechanism in addition to that of modulating transcriptional initiation. dam methylation, either directly or indirectly, inhibits the usage of IS50 sequences by the transposase. Thus, dam methylation can affect both the expression of the transposase and the DNA substrate upon which it acts.

lacZ translation initiation mutations

Sixteen single point mutations near the beginning of the lacZ gene have been isolated and their effect on lacZ expression has been measured. Five mutations were obtained that alter a potential stem-and-loop structure in the messenger RNA that masks the initiation codons. Formation of this stem-and-loop is a result of transcription of DNA sequences introduced during the cloning of the lac regulatory region. The mutations isolated were then moved into a background that deleted this structure. Analysis of these mutations indicated that the secondary structure inhibited lacZ expression 5.8-fold and that either single point mutations or a 9 base-pair deletion could relieve this inhibition completely. In addition, it was found that an A to C transversion in the first base following the initiation codon (in the absence of the inhibitory secondary structure) decreases lacZ expression almost twofold, whereas C to U transitions in the next two positions have negligible effects. Mutations were also obtained that either increase or decrease the length of the Shine-Dalgarno sequence. The effects of these mutations were studied in the presence or absence of the secondary structure that involves the two initiation codons. It was found that when translation initiation was inhibited by the secondary structure, increasing the length of the Shine-Dalgarno sequence increased lacZ expression 2.8-fold and decreasing the length of this sequence reduced lacZ expression 12-fold. When translation initiation was not inhibited by the secondary structure, increasing the length of the Shine-Dalgarno sequence had no effect and decreasing the length of this sequence only reduced lacZ expression sixfold. The mechanistic implications of these results are discussed. Two initiation codons are located in the beginning of the lacZ gene, 7 and 13 bases from the Shine-Dalgarno sequence. NH2-terminal sequence analysis indicated that the majority of the protein synthesized initiate at the first initiation codon in the wild-type lacZ gene (in agreement with results reported previously by J. L. Brown and his colleagues). Upon introduction of sequences that result in a change in the mRNA secondary structure, both initiation codons are used in almost equal amounts. Three mutations and two pseudorevertants were obtained, which are located in the first initiation codon. It was found that when the first initiation codon is changed from AUG to GUG, translation initiation is decreased tenfold at that codon.(ABSTRACT TRUNCATED AT 400 WORDS)