Transposase Family Research Articles

The Three-dimensional Structure of a Tn5Transposase-related Protein Determined to 2.9-Å Resolution

Transposon Tn5 employs a unique means of self-regulation by expressing a truncated version of the transposase enzyme that acts as an inhibitor. The inhibitor protein differs from the full-length transposase only by the absence of the first 55 N-terminal amino acid residues. It contains the catalytic active site of transposase and a C-terminal domain involved in protein-protein interactions. The three-dimensional structure of Tn5 inhibitor determined to 2.9-A resolution is reported here. A portion of the protein fold of the catalytic core domain is similar to the folds of human immunodeficiency virus-1 integrase, avian sarcoma virus integrase, and bacteriophage Mu transposase. The Tn5 inhibitor contains an insertion that extends the beta-sheet of the catalytic core from 5 to 9 strands. All three of the conserved residues that make up the "DDE" motif of the active site are visible in the structure. An arginine residue that is strictly conserved among the IS4 family of bacterial transposases is present at the center of the active site, suggesting a catalytic motif of "DDRE." A novel C-terminal domain forms a dimer interface across a crystallographic 2-fold axis. Although this dimer represents the structure of the inhibited complex, it provides insight into the structure of the synaptic complex.

Expression of the transposase gene tnpA of Tn4652 is positively affected by integration host factor.

Tn4652 is a derivative of the toluene degradation transposon Tn4651 that belongs to the Tn3 family of transposons (M. Tsuda and T. Iino, Mol. Gen. Genet. 210:270-276, 1987). We have sequenced the transposase gene tnpA of transposon Tn4652 and mapped its promoter to the right end of the element. The deduced amino acid sequence of tnpA revealed 96.2% identity with the putative transposase of Tn5041. Homology with other Tn3 family transposases was only moderate (about 20 to 24% identity), suggesting that Tn4652 and Tn5041 are distantly related members of the Tn3 family. Functional analysis of the tnpA promoter revealed that it is active in Pseudomonas putida but silent in Escherichia coli, indicating that some P. putida-specific factor is required for the transcription from this promoter. Additionally, tnpA promoter activity was shown to be modulated by integration host factor (IHF). The presence of an IHF-binding site upstream of the tnpA promoter enhanced the promoter activity. The positive role of IHF was also confirmed by the finding that the enhancing effect of IHF was not detected in the P. putida ihfA-deficient strain A8759. Moreover, the Tn4652 terminal sequences had a negative effect on transcription from the tnpA promoter in the ihfA-defective strain. This finding suggests that IHF not only enhances transcription from the tnpA promoter but also alleviates the negative effect of terminal sequences of Tn4652 on the promoter activity. Also, an in vitro binding assay demonstrated that both ends of Tn4652 bind IHF from a cell lysate of E. coli.

Retroviral integrase: conserved sequence information as a guide to understanding structure and function of the retroviral and bacterial IS3 DD(35)E transposases

The retroelement integrases (IN) are one of the three enzymes required for the retroviral life cycle. Much attention has been focused on retroviruses as a result of the AIDS epidemic and IN became a target for the rational development of anti-AIDS drug therapies. However two things that would help this effort, the mechanism of the chemical reaction catalysed by IN and the structure were initially unknown. This review shows how sequence alignments helped design and interpret experiments which showed that the conserved DDE residues were catalytically required, and which showed the catalytic function of three acidic residues in a motif here called the DD(35)E motif, in turn leading to the solution of its structure by xray crystallography; and finding the DNA binding function of the C-terminus in turn leading to the solution of its structure by NMR. Sequence alignments showed that IN belonged to a greater family of transposases found in retroelements and IS3 bacterial insertion sequences, whose function was to transpose a piece of dsDNA with the consensus sequence CA at the 3′-ends. IN was composed of three separate motifs. The central region of about 110 residues which very similar in sequence and which shared 3 conserved acidic groups (D,D and E). The 2nd D and the E were always separated by 35 residues, and together with the first D, were used to name this family the DD(35)E transposases. It was proposed that the DD(35)E region was an autonomously folding domain; had the catalytic site which recognised the CA 3′-ends; and that these 3 residues were involved in DNA bond breaking/making and divalent metal ion binding. The N-terminal part of IN contained a conserved HHCC motif, presumably a Zn finger motif, which was not found in the other DD(35)E transposases. Aside from the HHCC residues, this part of the enzymes did not align well suggesting that if the structure was conserved in the family, then the binding of the Zn atom would have to be the overriding factor. The C-terminus was very variable not able to be aligned within the DD(35)E transposase family, except for very closely related enzymes. The C-terminus for another transposase of similar size (γ–δres, Abdel-Meguid et al., Proc. Nat. Acad. Sci. USA 81, (1984) 2001–2005) was known to bind DNA. However no DNA binding motifs such as helix–turn–helix were found by computer assisted searches in the C-terminus of the IN family and no function was able to be assigned to it. It seemed likely that IN was a 3 domain protein and it was reasonable to explore the function of IN in domains in the same manner as had be pioneered with res.

Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor.

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.

A helix-turn-helix DNA-binding motif predicted for transposases of DNA transposons.

A helix-turn-helix (HTH) DNA-binding motif is identified in transposase sequences in Tc1, mariner and pogo DNA transposum. The findings are supported by results of various sequence analysis methods. Tc1 transposases are also predicted to contain another DNA-binding region. These findings are in accord with experimental evidence obtained from Tc1A, Tc3A and pogo transposases. The pogo family transposases, but not the pogo-type transcription factors, contain the HTH motif, suggesting that HTH structures are essential for Tc1/mariner/pogo transposition. Analysis of multiple sequence alignments enabled the identification of the HTH motif in distantly related protein sequences.

Tc1 Transposon-like Sequences are Widely Distributed in Salmonids

Transposon-like elements flanked by inverted repeats, although common in invertebrates, have only recently been found in vertebrates. We report the presence of Tc1 transposon-like sequences in salmon, trout and charr species and find that these elements belong to several families that do not follow phylogenetic lines. As many as 15,000 copies reside in the Atlantic salmon haploid genome. The complete DNA sequence of one of these transposon-like elements (SALT1) is 1535 base-pairs long, including 35 nucleotide-long terminal inverted repeats. It contains a degenerate open reading frame (ORF) of 1273 nucleotides whose inferred amino acid sequence shares sequence similarity with the "D,D35E" family of transposases, particularly those from Caenorhabditis sp. and Drosophila sp. Southern blot analysis indicated that Tc1 transposon-like sequences are present in other lower vertebrates, including several fish species and amphibians, but the copy number can vary significantly in different lineages.

Nucleotide Sequence Analysis of IS1533 from Leptospira borgpetersenii: Identification and Expression of Two IS-Encoded Proteins

The nucleotide sequence of IS1533, an insertion sequence-like element cloned from the spirochete Leptospira borgpetersenii, was determined. IS1533 contains imperfect terminal inverted repeats (IVR) of 31 bp flanking a 1402-bp internal sequence. A putative target sequence was identified, and insertion may result in duplication of 2 bp. The internal sequence has a single open reading frame (ORF). IS1533 encodes two proteins (43.5 and 41 kDa) initiating alternatively at either the first or the second AUG codons of the ORF. These proteins are related to a recently recognized family of IS-encoded transposases and bacterial recombinases, all which share a region of homology with the active site of the HIV reverse transcriptase. The IS1533-encoded proteins were expressed in Escherichia coli . Both the 43.5- and 41-kDa proteins bound IS1533 DNA probes in a Southwestern blot assay. These data suggest that one or both proteins function during transposition of IS1533.

Structures of the Euplotes crassusTec1 and Tec2 elements: identification of putative transposase coding regions

The Tec 1 and Tec 2 transposon-like element families of Euplotes crassus are highly unusual in that all 30 000 copies of each family are excised from the genome during a discrete time period of macronuclear development. Complete nucleotide sequences were generated for the Tec 1-1 and Tec 2-1 elements, representing the Tec 7 and Tec 2 families. Open reading frames (ORFs) are conserved in position and sequence between the two elements, although sequences that comprise one ORF (ORF2) of Tec 1-1 are split into two overlapping ORFs (ORFs 2A and 2B) in Tec 2-1. ORF1 in Tec 1-1, its homolog in Tec 2-1 and one of the overlapping ORFs from Tec 2-1 (ORF2B) contain TGA codons, which may be translated as Cys, as observed for two other Euplotid genes. Sequence analyses of ORFs from other members of each element family indicate that the families are distinct from each other and are highly conserved within each family. Computer searches of sequence databases have revealed sequence similarity between Tec ORF 1s and the previously described Tc 1-IS 630 family of transposases which includes ORFs from bacterial, nematode and insect transposons.