TdIF1 recognizes a specific DNA sequence through its Helix-Turn-Helix and AT-hook motifs to regulate gene transcription

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TdIF1 promotes transcription in a luciferase reporter assay.

<p>(A) TdIF1 promotes transcription. The pGL3-promoter-TdIF1-binding sequence and pRL-TK were co-transfected with pEGFP or pEGFP-TdIF1 into 293T cells, and the luciferase activity was assayed. The relative luciferase activity was normalized to the value obtained with the pGL3-promoter and pEGFP. Error bars represent S.E.M. Samples significantly different from the control are indicated by asterisks (p<0.01). (B) TdIF1 promotes transcription by directly binding to DNA. The pGL3-promoter-TdIF1-binding sequence and pRL-TK were co-transfected with pEGFP-TdIF1 truncated and point mutants, and the luciferase activity was assayed. Error bars represent S.E.M.</p

Structural basis for toughness and flexibility in the C-terminal passenger domain of an acinetobacter trimeric autotransporter adhesin

Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three β-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner β-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions. © 2016 American Society for Biochemistry and Molecular Biolog

Identification of the DNA sequences recognized by TdIF1 by SELEX.

<p>(A) Flow chart of the SELEX experiment. (B) EMSA results in the 7th cycle. DNA was detected by EtBr staining. The arrow indicates the TdIF1/oligoDNA complex. (C) Oligonucleotides selected by SELEX aligned around the identified consensus 5′-GNTGCATG-3′, underlined in each case. Sequences of six or more continuous As or Ts are in red. Flanking linker sequence shown in italics. (D) Conserved nucleotides drawn as a sequence logo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066710#pone.0066710-Crooks1" target="_blank">[20]</a>.</p

TdIF1 preferentially recognizes 5′-GNTGCATG-3′ following an AT-tract.

<p>(A) Sequences of the biotin-labelled AT-rich probe and the competitors used. Asterisks indicate replaced residues in the AT-tract or 5′-GNTGCATG-3′ motifs. (B) Competitive EMSA. All reaction mixtures contained 5 pmol of biotin-labelled AT-rich probe. Purified His-TdIF1 (100 ng) was incubated in the reaction mixture alone or with competitors (5, 15, or 45 pmol) as indicated (lanes 2–14). (C) Competitive EMSA using TdIF1mtHTH2, which has mutations in the HTH. TdIF1mtHTH2 (200 ng) or wild-type TdIF1 (100 ng) was incubated with biotin-labelled AT-rich probe alone or with competitors (15 or 45 pmol), as indicated.</p

Ubiquitylation of Terminal Deoxynucleotidyltransferase Inhibits Its Activity

<div><p>Terminal deoxynucleotidyltransferase (TdT), which template-independently synthesizes DNA during V(D)J recombination in lymphoid cells, is ubiquitylated by a BPOZ-2/Cul3 complex, as the ubiquitin ligase, and then degraded by the 26 S proteasome. We show here that TdT is ubiquitylated by the Cul3-based ubiquitylation system <em>in vitro</em>. Because TdT could also be ubiquitylated in the absence of Cul/BPOZ-2, we determined that it could also be directly ubiquitylated by the E2 proteins UbcH5a/b/c and UbcH6, E3-independently. Furthermore, the ubiquitylated TdT inhibited its nucleotidyltransferase activity.</p> </div

UbcH5a and UbcH6 promote TdT ubiquitylation.

<p>(A) UbcH5a enhances TdT ubiquitylation in 293 T cells. 293 T cells were transfected with plasmids encoding His-Ub (lanes 1, 3 to 5), Myc-TdT (lanes 2 to 5), and/or Flag-UbcH5a (lanes 1, 2, 4 and 5) in the indicated combinations. After a 24 h incubation, the cells were treated with 10 µM MG132 for another 12 h, lysed under denaturing conditions, and the ubiquitylated proteins were affinity-purified and separated by SDS-PAGE. Ubiquitylated Myc-TdT was detected by immunoblotting with an anti-Myc antibody. Myc-TdT and Flag-UbcH5a in the lysate were detected using an anti-Myc or anti-Flag antibody. (B) UbcH6 enhances TdT ubiquitylation in 293 T cells. 293 T cells were transfected with plasmids encoding His-Ub (lanes 1, 3 to 5), Flag-TdT (lanes 2 to 5), and/or Myc-UbcH6 (lanes 1, 2, 4 and 5) in the indicated combinations. After incubation for 24 h, the cells were treated with 10 µM MG132 for another 12 h. The cells were lysed under denaturing conditions, and the ubiquitylated proteins were affinity-purified and separated by SDS-PAGE. Ubiquitylated TdT was detected by immunoblotting using an anti-Flag antibody. Flag-TdT and Myc-UbcH6 in the lysate were detected using an anti-Flag or anti-Myc antibody. (C) UbcH7 does not enhance TdT ubiquitylation in 293 T cells. 293 T cells were transfected with plasmids encoding His-Ub (lanes 1, 3 to 5), Flag-TdT (lanes 2 to 5), and/or Myc-UbcH7 (lanes 1, 2, 4 and 5) in the indicated combinations. After a 24 h incubation, the cells were treated with 10 µM MG132 for another 12 h, lysed under denaturing conditions, and the ubiquitylated proteins were affinity-purified and separated by SDS-PAGE. Ubiquitylated TdT was detected by immunoblotting using an anti-Flag antibody. Flag-TdT and Myc-UbcH7 in the lysate were detected using an anti-Flag or anti-Myc antibody.</p

Knockdown of UbcH5, UbcH6, or UbcH7 mRNA reduces TdT ubiquitylation.

<p>(A and B) 293 T cells were transfected with a control siRNA or siRNAs targeting UbcH5a or UbcH5c using the X-tremeGENE Transfection reagent 24 h prior to the plasmid transfection. The cells were then transfected with Myc-TdT and His-Ub using the GeneJuice Transfection Reagent. After a 24 h incubation, the cells were treated with 10 µM MG132 for another 12 h. mRNA levels of UbcH5 isoforms were measured by real-time PCR, normalized to GAPDH, and are expressed as their ratio to cells transfected with a control siRNA (A). Ubiquitylated Myc-TdT and UbcH5 in the lysate were detected by immunoblotting with an anti-Myc, anti-UbcH5, or anti-actin antibody (B). (C) 293 T cells were transfected with a control siRNA or siRNAs targeting UbcH6 or UbcH7 using the X-tremeGENE Transfection reagent 24 h prior to the plasmid transfection. The cells were then transfected with Myc-TdT and His-Ub using the GeneJuice Transfection Reagent. After incubation for 24 h, the cells were treated with 10 µM MG132 for another 12 h. Ubiquitylated Myc-TdT, UbcH6, and UbcH7 in the lysate were detected by immunoblotting with an anti-Myc, anti-UbcH6, anti-UbcH7, or anti-actin antibody.</p

TdT ubiquitylation <i>in vivo</i> or <i>in vitro</i>.

<p>(A) Endogenous TdT ubiquitylation in Jurkat cells. Immunoprecipitation was carried out using an anti-TdT antibody (lanes 4 and 6) or rabbit pre-immune serum (pre-IS) (lanes 3 and 5). Immunoprecipitants were subjected to SDS-PAGE and immunoblotted with anti-Ub (FK2) or anti-TdT antibody. One mg of whole cell lysate was used for each immunoprecipitation. (B) <i>In vitro</i> TdT ubiquitylation. The substrate His-TdT was incubated under ubiquitylation conditions with purified proteins. His-TdT and His-BPOZ-2 were purified from <i>E. coli</i> and the Flag-tagged proteins were purified from 293 T cells. To obtain Cul3/Rbx1 or BPOZ-2/Cul3/Rbx1 complexes, 293 T cells were co-transfected with the circled plasmids (lanes 10 and 11). After electrophoresis with a denaturing 7.5% polyacrylamide gel, ubiquitylated TdT was detected using an anti-TdT antibody. (C and D) Kinetics of TdT ubiquitylation in the absence (C) or presence (D) of the BPOZ-2/Cul3/Rbx1 complex. The incubation time after the addition of His-TdT is given above. The reaction products were separated by 7.5% SDS-PAGE and immunoblots were probed with anti-TdT antibody or anti-Flag antibody. The ratio of ubiquitylated His-TdT to unmodified His-TdT was determined with ImageJ. The band density for ubiquitylated His-TdT was defined as 100%.</p