7 research outputs found

    Location of insertions within the amino acid sequence and relative transposition activity of each mutant.

    No full text
    <p>Amino acid positions are numbered under the sequence. Secondary structures (determined with DSSP, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922-Kabsch1" target="_blank">[74]</a>) are indicated under the corresponding sequence as bars (α-helices) or arrows (β-strands). Small vertical arrows point to the exact location of each insertion (in each three-letter amino acid code, the first letter represents the first nucleotide of the corresponding codon, etc.), and the attached coloured boxes indicate ranges of transposition activity of each mutant relative to the wild type activity (colors denoting the percentage ranges are shown at the lower-right). The number within the boxes indicate the observed relative activity.</p

    Identification of insertion-tolerant regions in MuA on the basis of pentapeptide insertion analysis and alignment-based data.

    No full text
    <p>Above the amino acid sequence are shown with arrows the pentapeptide-insertion tolerant sites (activity level ≥1%) colour-coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone-0037922-g004" target="_blank">Figure 4</a>. The percentage range chart is shown at the lower-right. Two or three arrows per site are indicative of insertions involving more than one reading frame. Below the amino acid sequence are shown the secondary structural elements (arrows and rectangles). The elements are connected with line segments indicating the length of each PDB structure. Below the structural elements are shown the subdomains as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone-0037922-g003" target="_blank">Figure 3</a>. Above the bolded line, the downward black and upward white arrows represent the alignment-based insertion and deletion (indel) data, respectively (each particular indel precedes the marked amino acid). The maximum indel length at each site is indicated by a number shown above each arrow (data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922.s001" target="_blank">Figure S1</a>). The stars indicate the DDE-motif residues (D269, D336, E392).</p

    Mu transpososome architecture at post-integration stage and MuA regions allowing pentapeptide insertions.

    No full text
    <p>The overall organization is sketched according to the unpublished crystallographic structure of Mu transpososome (P. Rice and S.P. Montaño, personal communication). The structure constitutes an essential framework for a meaningful interpretation of the functional data (see Discussion). Transposon end segments are shown with black lines, MuA binding sites (R1 and R2) are highlighted with rectangles, and the attached target DNA is shown in magenta. The catalytic MuA protomers are shown in orange and the non-catalytic protomers in brown. Insertion-tolerant subdomains are highlighted with red. The arrows (shown only for one protomer) indicate those linker and loop regions, in which insertions are tolerated well (wild type protein activity retained). The catalytic protomers act <i>in trans</i>, i.e. the protomer bound to one end catalyzes DNA cleavage and joining reactions in the other end.</p

    Assembly and function of Mu transpososome core.

    No full text
    <p>This pathway is based on <i>in vitro</i> studies and utilizes a minimum number of macromolecular components <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922-Savilahti1" target="_blank">[27]</a>. The <i>in vivo</i> assay described earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922-Pajunen3" target="_blank">[60]</a> and used here (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922.s001" target="_blank">Figure S1</a>) is a close mimic of this minimal-component <i>in vitro</i> system with regard the following features: (i) the configuration includes two MuR-ends (with R1 and R2 MuA binding sites), (ii) the phage-encoded MuB protein and (iii) transpositional enhancer are not included. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922.s001" target="_blank">Figure S1</a> for a full description of Mu transposition pathway and its comparison to the pathway used in the papillation analysis. The R1 and R2 MuA binding sites are shown as rectangles. MuA is drawn as a tetramer of yellow circles and target DNA is shown in purple. The small arrows on the target DNA indicate the 5-bp staggered locations for strand transfer on the two strands. The dots in the assembled transpososome indicate the Mu end cleavage sites.</p

    RNaseH fold of MuA encompassing the amino acid region E258–G396.

    No full text
    <p>(A) The conserved secondary structural elements, the central β-sheet (with numbered strands 1–5) and the adjacent α-helix, are shown with light blue and light green, respectively. The DDE-motif residues are shown in yellow with exposed sidechains. Pentapeptide-insertion-tolerant sites G282, G329, and R355 are colour-coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone-0037922-g004" target="_blank">Figure 4</a>. (B) Mapping of the alignment-based insertions and deletions. The orange-coloured amino acids depict insertion sites, and the accompanying numbers identify the respective amino acids (insertions occur between the specified residues). Maximum length deletions are coloured with magenta and indicated with respective amino acid residue numbers.</p

    MuA domain structure and location of pentatapeptide insertions with respective transposase activities.

    No full text
    <p>(A) Structural organization of MuA with different functions assigned to various subdomains. The numbers correspond to the amino terminus of each subdomain as specified earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922-Schumacher1" target="_blank">[47]</a>. (B) Enzymatic activities of MuA variants plotted against the 5 aa insertion site of each respective mutant protein. Activities obtained from the papillation assay are presented as a percentage of the wild type activity. For each protein variant the mean from three replicates is shown.</p

    Papillation assay.

    No full text
    <p>(A) Phenotypically Lac<sup>– </sup><i>E. coli</i> strain is transformed with a plasmid carrying a reporter transposon and encoding arabinose-inducible <i>MuA</i> transposase gene. Following expression of MuA, the reporter transposon is mobilized. Transposition into an expressed gene (<i>geneX</i>) in the correct orientation and reading frame generates a <i>geneX::lacZ</i> gene fusion, expressing a protein fusion with a C-terminal β-galactosidase moiety. Such events can be detected as blue papillae in bacterial colonies growing on Xgal-containing indicator plates. This quantitative assay directly measures the activity of the MuA variant analyzed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922-Pajunen3" target="_blank">[60]</a>. (B) Colonies from papillation assay. Shown are colonies representing one hypoactive MuA variant (clone #188, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922.s003" target="_blank">Table S1</a>), wild type MuA, and one hyperactive MuA variant (clone #170, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037922#pone.0037922.s003" target="_blank">Table S1</a>). Three representative colonies per variant are shown, indicating a high degree of reproducibility.</p
    corecore