Identification of Translin and Trax as Components of the GS1 Strand‐Specific DNA Binding Complex Enriched in Brain

Abstract

In previous gel-shift assays, we identified a protein complex, referred to as GS1, that binds in a sequence-specific manner to single-stranded DNA and is highly enriched in brain. As an initial step in clarifying the function of this complex, we have undertaken studies aimed at defining its protein components. In particular, we focused on identifying two protein bands that were covalently labeled when the GS1 -DNA complex was subjected to UV irradiation to induce cross-linking between the radiolabeled probe and GS1 components. By following GS1 binding activity through a series of conventional chromatographic steps, as well as an affinity column based on the DNA oligonucleotide used for gel-shift assays, we were able to achieve -500,000-fold enrichment of GS1 compared with that in crude cerebellar extracts used as starting material. This highly purified fraction contained both protein bands detected by UV crosslinking in crude extracts. Sequencing of peptides derived from these proteins led to their identification as Translin and Trax, interacting proteins identified in studies of DNA recombination in lymphocytes. A distinct line of research has provided evidence that a complex containing Translin can bind to specific mRNAs and block their translation. Whether one or both of these proposed functions of the Translin/Trax complex explains the high basal level of GS1 binding activity present in the brain remains to be determined. Key Words: Translin-Trax-GS1 protein complex-DNA recombination-Lymphocyte-mRNA translation-GS1 binding activity-Brain.

Part of Xenopus Translin Is Localized in the Centrosomes during Mitosis

“…The gel band corresponding to p35 was in-gel digested, treated as described under Materials and Methods and submitted to nanoelectrospray MS and MS/MS spectra on a Qtof mass spectrometer. From the recorded MS/MS mass spectra four partial sequences were found: XXF NCBInr data banks search revealed a high degree of homology with human, rat and chicken Trax, a previous described Translin-associated protein which has been suggested to be involved in the active nuclear transport of Translin (22,23,25) (Fig. 3C).…”

“…Native form of Translin has been described as a ring-shaped structure with an assembly of eight subunits of 27 kDa monomer whose molecular mass is approximately 220 kDa (21). Moreover, several studies have shown that TB-RBP/Translin can interact with different proteins such as Trax, ␥-Actin, and the transitional endoplasmic reticulum ATPase (22)(23)(24)(25). To investigate if X-translin is present in metaphase II-arrested oocytes in association with other proteins, we have developed a gel filtration analysis.…”

Trax (Translin-associated Factor X), a Primarily Cytoplasmic Protein, Inhibits the Binding of TB-RBP (Translin) to RNA

“…Sequence analysis in a meiotic recombination hot spot region of human chromosome 16 shows TB-RBP/Translin binding sequences near the breakpoint (7). TB-RBP/Translin has also been proposed to act as a single-stranded DNA-binding transcription factor, which activates early response gene expression in the brain (8). TB-RBP/Translin also functions as a RNA-binding protein mediating intracellular and intercellular mRNA transport (9,10).…”

“…Both have putative leucine zipper domains at the C terminus of TB-RBP and in the mid-region of Trax (3,7). Interaction between TB-RBP and Trax has been demonstrated by coimmunoprecipitation (19) and in the yeast two-hybrid assay (7,8). Yeast two-hybrid and in vitro binding studies indicate that TB-RBP dimers are the minimum unit needed for DNA or RNA binding (20).…”

Genomic structure and chromosomal localization of the gene encoding TRAX, a Translin-associated factor X

“…The TRAX protein contains bipartite nuclear targeting sequences in its N-terminal region (Aoki et al 1997a), suggesting a possible role in the selective nuclear transport of Translin protein lacking any nuclear targeting motifs. It has been reported that TRAX and Translin associate in vivo as components of the NS1 strand-specific DNA binding complex enriched in brain (Taira et al 1998). Because Translin was found as a homopolymer in lymphoid cells, these results raise the possibility that Translin forms heteropolymers with TRAX in the central nervous system and regulates cellular functions, reminiscent of observations with Bcl-2-Bax (Oltvai et al 1993) or Myc-Max interactions (Blackwood and Eisenman 1991).…”