The TFIID pivot of preinitiation complex

Science China Life Sciences -     

Nuclear factor I enhances adenovirus DNA replication by increasing the stability of a preinitiation complex.

Nuclear factor I (NFI) or its isolated DNA-binding domain (NFI-BD) enhances initiation of adenovirus DNA replication up to 50-fold at low concentrations of the precursor terminal protein-DNA polymerase (pTP-pol) complex. Both in solution and when bound to DNA, NFI-BD can form a complex with pTP-pol. To investigate the mechanism of enhancement by NFI, we determined the stability of a functional preinitiation complex formed in vitro between pTP-pol and the origin. Challenge experiments with a distinguishable template containing an identical origin revealed that in the absence of NFI, this preinitiation complex was very sensitive to competition for pTP-pol. Addition of NFI-BD increased the half-life of the complex at least 10-fold and led to the formation of a template-committed preinitiation complex. In agreement with this, binding of pTP-pol to origin DNA in band-shift assays was enhanced by NFI. By DNase I footprinting we show that the specificity of binding as well as induction of structural changes in origin DNA by pTP-pol are increased by NFI. These results indicate that NFI, by binding and positioning pTP-pol, stabilizes the complex between pTP-pol and the core origin, and thus enhances initiation of DNA replication.     

Efficient incorporation of eukaryotic initiation factor 1 into the multifactor complex is critical for formation of functional ribosomal preinitiation complexes in vivo

Eukaryotic initiation factor 1 (eIF1) is a low molecular weight factor critical for stringent AUG selection in eukaryotic translation. It is recruited to the 43 S complex in the multifactor complex (MFC) with eIF2, eIF3, and eIF5 via multiple interactions with the MFC constituents. Here we show that FLAG epitope tagging of eIF1 at either terminus abolishes its in vitro interactions with eIF5 and eIF2beta but not that with eIF3c. Nevertheless, both forms of FLAG-eIF1 fail to bind eIF3 and are incorporated into the 43 S complex inefficiently in vivo. C-terminal FLAG tagging of eIF1 is lethal; overexpression of C-terminal FLAG-eIF1 severely impedes 43 S complex formation and derepresses GCN4 translation due to limiting of eIF2.GTP.Met-tRNA(i)(Met) ternary complex binding to the ribosome. Furthermore, N-terminal FLAG-eIF1 overexpression reduces eIF2 binding to the ribosome and moderately derepresses GCN4 translation. Our results provide the first in vivo evidence that eIF1 plays an important role in promoting 43 S complex formation as a core of factor interactions. We propose that the coordinated recruitment of eIF1 to the 40 S ribosome in the MFC is critical for the production of functional 40 S preinitiation complex.