Protein interactions involved in tRNA gene-specific integration of Dictyostelium discoideum non-long terminal repeat retrotransposon TRE5-A

Abstract

Mobile genetic elements that reside in gene-dense genomes face the problem of avoiding devastating insertional mutagenesis of genes in their host cell genomes. To meet this challenge, some Saccharomyces cerevisiae long terminal repeat (LTR) retrotransposons have evolved targeted integration at safe sites in the immediate vicinity of tRNA genes. Integration of yeast Ty3 is mediated by interactions of retrotransposon protein with the tRNA gene-specific transcription factor IIIB (TFIIIB). In the genome of the social amoeba Dictyostelium discoideum, the non-LTR retrotransposon TRE5-A integrates approximately 48 bp upstream of tRNA genes, yet little is known about how the retrotransposon identifies integration sites. Here, we show direct protein interactions of the TRE5-A ORF1 protein with subunits of TFIIIB, suggesting that ORF1p is a component of the TRE5-A preintegration complex that determines integration sites. Our results demonstrate that evolution has put forth similar solutions to prevent damage of diverse, compact genomes by different classes of mobile elements.

FIG. 1.

DdTFIIIB subunits. (A) Clustal X alignment of DdTBP with HsTBP and ScTBP. The 22.8-kDa DdTBP has 69% and 68% sequence identity with the conserved carboxy-terminal part of its yeast and human orthologs, respectively. Exposed α-helices along the convex surface of TBP are indicated in boxes. DdTBP is encoded by gene tbpA and is listed under Dictybase entry DDB0185066 (http://dictybase.org/). (B) Identification of a DdBrf1 ortholog. Brf1 proteins can be divided into two functional domains: the conserved TFIIB-like amino-terminal domain and highly divergent carboxy-terminal domains. Clustal X alignment of the conserved TFIIB-homologous region of DdBrf1 (DdBrf^1-270) with corresponding regions of HsBrf1 and ScBrf1. The TFIIB-like domain shows 42% and 48% sequence identity with the corresponding regions in ScBrf1 and HsBrf1, respectively. DdBrf1 is 706 amino acids in length (79.9 kDa) and is encoded by gene brf1 (Dictybase entry DDB0204087). (C) Identification of a Bdp1 ortholog in the D. discoideum genome. Use of full-length ScBdp1 as the query sequence in Blastp searches (1) identified a protein that could be aligned within a central domain spanning 130 amino acids with corresponding regions of Schizosaccharomyces pombe Bdp1 (SpBdp1) and ScBdp1 and the human ortholog (HsBdp1). The SANT domain (17) is boxed. DdBdp1 is encoded by gene bdp1 (Dictybase entry DDB0220508).

FIG. 2.

DdTFIIIB subunits interact with TRE5-A ORF1p. (A) Model for initiation of tRNA gene transcription in D. discoideum. DdTFIIIC binds to the tRNA gene-internal B box sequence and then recruits DdTFIIIB. The latter consists of the three subunits DdTBP, DdBrf1, and DdBdp1 and occupies space at the 5′ end of the tRNA gene close to the TRE5-A integration site. (B) DdTFIIIB subunits were tested for interaction with TRE5-A-derived ORF1p and ORF2p proteins. Selection plates contained minimal medium without histidine that were supplemented with either 5 mM 3-AT (left) or 6 mM 3-AT (right). Combinations of two-hybrid vectors are listed; the left column represents proteins expressed from pBT vectors, and the right column lists proteins cloned into the pTRG vector. (C) Schematic presentation of full-length TRE5-A.1 and the derived proteins tested in two-hybrid assays. The ORF2 protein was cloned in three parts according to the predicted ENp and RTp domains and the HCp domain of unknown function. Modules A, B, and C represent 5′ and 3′ untranslated regions of TRE5-A.1. (D) Expression of pBT-derived fusion proteins in the bacterial two-hybrid reporter strain. Western blot showing expression levels of DdTFIIIB subunits and TRE5-A-encoded ORF proteins. The corresponding DNA fragments, cloned in vector pBT, were transformed into the BacterioMatch II reporter strain. Bacteria were grown at 30°C. Extracts were prepared from whole cells and subjected to SDS-PAGE on 10% polyacrylamide gels. After electrophoretic transfer onto nitrocellulose membranes, proteins were stained with a commercial polyclonal antiserum directed against the λcI repressor protein encoded in the pBT vector (Stratagene). Lanes 1 and 2, DdTBP; lanes 3 and 4, DdBrf^1-270; lanes 5 and 6, DdBrf^271-706; lanes 7 and 8, DdBdp1; lanes 9 and 10, ORF1p; lanes 11 and 12, ENp; lanes 13 and 14, RTp; lanes 15 and 16, HCp. Lanes 1, 3, 5, 7, 9, 11, 13, and 15 show extracts of bacteria without induction of recombinant protein expression. In lanes 2, 4, 6, 8, 10, 12, 14, and 16, expression of recombinant proteins was induced with 0.5 mM IPTG for 3 h at 30°C. Asterisks denote the bands corresponding to the molecular masses of the full-length λcI fusion proteins. (E) Pull-down experiment. ³⁵S-labeled DdTBP or full-length DdBrf1 was incubated with bacterially expressed MBP or an MBP-ORF1p fusion protein. Bound DdTBP or DdBrf1 was subjected to SDS-PAGE followed by autoradiography. Input refers to 10% of a labeling reaction.

FIG. 3.

Determination of protein interaction domains on ORF1p. (A) Two-hybrid assays were performed to determine binding domains for DdTBP and DdBrf1 on ORF1p. The selection plates contained minimal medium without histidine that was supplemented with 5 mM 3-AT. Combinations of two-hybrid vectors are listed; the left column represents proteins expressed from pBT vectors, and the right column lists proteins cloned into the pTRG vector. (B) Presentation of ORF1p deletions tested for interaction with ORF1p (dimerization), DdTBP, and DdBrf1. + and − refer to growth of transformants on selection plates containing 5 mM 3-AT.

FIG. 4.

Determination of protein binding sites on DdTBP. (A) The crystal structure of HsTBP was used as a template to model DdTBP (see Materials and Methods). The α-helices along the convex surface of TBP are indicated. The N and C termini of the protein are also indicated. (B) Two-hybrid assays for the interaction of DdTBP mutants with DdBrf1 (left) and ORF1p (right). Selections were performed on plates containing 5 mM 3-AT. Combinations of two-hybrid vectors are listed; the left column represents proteins expressed from pBT vectors, and the right column lists proteins cloned into the pTRG vector. (C) Delineation of amino acid positions of α-helices H1, H2, H1′, and H2′ in DdTBP (total length, 205 amino acids). (D) Amino acid sequences of the carboxy termini of DdTBP and HsTBP. The α-helix H2′ is written in uppercase letters. The arrow depicts the S195A and S195K mutations in DdTBP tested in the two-hybrid experiments.

FIG. 5.

TRE5-A ORF1p binds to an HsTBP/DdTBP chimera. (A) Modeled structure of HsTBP (light gray) containing the α-helix H2′ sequence of DdTBP (black). Variant amino acid positions in HsTBP and DdTBP helix 2H′ are written in bold. The amino acid sequence of the carboxy terminus in the chimeric HsTBP[DdH2′] protein is indicated. (B) Two-hybrid experiment showing the binding of HsTBP[DdH2′] to ORF1p and DdBrf1. Combinations of two-hybrid vectors are listed; the left column represents proteins expressed from pBT vectors, and the right column lists proteins cloned into the pTRG vector. Selections were performed on plates containing 3 mM 3-AT.

FIG. 6.

Clustal X alignment of ORF1 proteins from D. discoideum TRE5-A and TRE5-B retrotransposons. The DdTBP-interacting region defined in TRE5-A ORF1p is boxed.

FIG. 7.

TRE5-B ORF1p binds to TFIIIB subunits. A two-hybrid experiment with TRE5-B ORF1p (cloned in pTRG) and the DdTFIIIB subunit proteins (cloned in pBT). The plate contained histidine-free medium supplemented with 5 mM 3-AT. Combinations of two-hybrid vectors are listed; the left column represents proteins expressed from pBT vectors, and the right column lists proteins cloned into the pTRG vector.