TATA binding protein (TBP) is a key regulator of RNA polymerase transcription. It binds to core promoters, often in large multiprotein complexes, and nucleates RNA polymerase II (Pol II) transcription initiation. In addition to the previously described TBP-like factor present in metazoans (TLF/TRF2/TRP/TLP), we describe a third, vertebrate-specific member of the TBP protein family from zebrafish, called TBP2. Evolutionary conserved TBP2 homologs were also found in human, mouse, frog, and pufferfish. The N-terminal domains of TBP2s are divergent amongst themselves and different from those of TBPs; however, the core domain of TBP2s and TBPs are almost identical. TBP2 binds the TATA box, interacts with TFIIA and TFIIB (similarly to TBP), and can mediate Pol II transcription initiation. However, TBP2 shows contrasting expression patterns in the gonads and during embryonic development in comparison to TBP, suggesting differential function. Knockdown of zebrafish TBP2 results in specific reduction of the protein level, leading to a phenotype, which indicates the requirement of TBP2 for embryonic patterning. The presence of three different TBP family members in vertebrates suggests the existence of developmental stage- and tissue-specific preinitiation complexes with specific requirements for different TBP family members.

Nucleoporin Nsp1p, which has four predicted coiled-coil regions (coils 1 to 4) in the essential carboxyterminal domain, is unique in that it is part of two distinct nuclear pore complex (NPC) subcomplexes, Nsp1p-Nup57p-Nup49p-Nic96p and Nsp1p-Nup82p-Nup159p. As shown by in vitro reconstitution, coiled-coil region 2 (residues 673 to 738) is sufficient to form heterotrimeric core complexes and can bind either Nup57p or Nup82p. Accordingly, interaction of Nup82p with Nsp1p coil 2 is competed by excess Nup57p. Strikingly, coil 3 and 4 mutants are still assembled into the core Nsp1p-Nup57p-Nup49p complex but no longer associate with Nic96p. Consistently, the Nsp1p-Nup57p-Nup49p core complex dissociates from the nuclear pores in nsp1 coil 3 and 4 mutant cells, and as a consequence, defects in nuclear protein import are observed. Finally, the nsp1-L640S temperature-sensitive mutation, which maps in coil 1, leads to a strong nuclear mRNA export defect. Thus, distinct coiled-coil regions within Nsp1p-C have separate functions that are related to the assembly of different NPC subcomplexes, nucleocytoplasmic transport, and incorporation into the nuclear pores.The nuclear pore complex (NPC), a structural entity conserved throughout evolution, spans the nuclear membranes and thus allows exchange of molecules between the cytoplasm and nucleus (for a review, see reference 43). Its octagonal symmetry is reflected on every substructure observed in electron microscopy. Two ring-like structures consisting of eight globular units are attached to the inner and outer parts of the nuclear membrane. While the cytoplasmic ring carries short filamentous protrusions, the nuclear ring extends into a basketlike structure. Together with a central structural framework of eight spokes, both rings form a channel for the signal-and energy-dependent nucleocytoplasmic transport of molecules (15,34).Based on the recent analysis of isolated yeast NPCs, it is expected that only about 30 individual proteins are required to build a nuclear pore complex (35). Most of these nucleoporins (Nups) had been identified before by using either genetic or biochemical approaches (for reviews, see references 9 and 36).

Nup116p is a GLFG nucleoporin involved in RNA export processes. We show here that Nup116p physically interacts with the Nup82p-Nsp1p-Nup159p nuclear pore subcomplex, which plays a central role in nuclear mRNA export. For this association, a sequence within the Cterminal domain of Nup116p that includes the conserved nucleoporin RNA-binding motif was sufficient and necessary. Consistent with this biochemical interaction, protein A-Nup116p and the protein A-tagged Nup116p C-terminal domain, like the members of the Nup82p complex, localized to the cytoplasmic side of the nuclear pore complex, as revealed by immunogold labeling. Finally, synthetic lethal interactions were found between mutant alleles of NUP116 and all members of the Nup82p complex. Thus, Nup116p consists of three independent functional domains: 1) the C-terminal part interacts with the Nup82p complex; 2) the Gle2p-binding sequence interacts with Gle2p/Rae1p; and 3) the GLFG domain interacts with shuttling transport receptors such as karyopherin-␤ family members.The nuclear pore complex (NPC) 1 is a huge organelle with an intricate structure of octagonal symmetry (for review, see Ref.

We provide evidence that a prokaryotic insertion sequence (IS) element is active in a vertebrate system. The transposase of Escherichia coli element IS30 catalyzes both excision and integration in extrachromosomal DNA in zebra¢sh embryos. The transposase has a pronounced target preference, which is shown to be modi¢ed by fusing the enzyme to unrelated DNA binding proteins. Joining the transposase to the cI repressor of phage V V causes transposition primarily into the vicinity of the V V operator in E. coli, and linking to the DNA binding domain of Gli1 also directs the recombination activity of transposase near to the Gli1 binding site in zebra¢sh. Our results demonstrate the possibility of fusion transposases to acquire novel target speci¢city in both prokaryotes and eukaryotes. ß