Interaction of Escherichia coli RNA Polymerase with the Eukaryotic TATA-Binding Protein

Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its subunit. Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of a ³²P-labeled phosph-amide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme–oligonucleotide covalent complex decreased with increasing TBP concentration. This was considered as indirect evidence for complexing of RNA polymerase with TBP. In gel retardation assays, the holoenzyme, but neither the minimal enzyme nor the subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the subunit. It was assumed that E. coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme.     

Porcine Type III RNA Polymerase III Promoters for Short Hairpin RNA Expression

Based on the highly conserved sequences of small nuclear RNA and small cytoplasmic RNA between vertebrate species, three porcine type III RNA polymerase III promoters, pY1, pY3 and pU6, were identified by using genomic DNA walking. To test the functional relationship of these sequences, the human H1 promoter of pSUPER-EGFP-l-neo vector was substituted with these three promoters to create the ppPol III-MCS vectors. The strength of each promoter was measured by its ability to derive expression of shRNA to repress expression of luciferase via RNA interference in the pig kidney epithelial cell line LLC-PK1. We determine that the ranking of promoter strength in descending order is pU6 > pY1 > pY3.     

Inhibition of TATA binding protein dimerization by RNA polymerase III transcription initiation factor Brf1

The Brf1 subunit of TFIIIB plays an important role in recruiting the TATA-binding protein (TBP) to the up-stream region of genes transcribed by RNA polymerase III. When TBP is not bound to promoters, it sequesters its DNA binding domain through dimerization. Promoter assembly factors therefore might be required to dissociate TBP into productively binding monomers. Here we show that Saccharomyces cerevisiae Brf1 induces TBP dimers to dissociate. The high affinity TBP binding domain of Brf1 is not sufficient to promote TBP dimer dissociation but in addition requires the TFIIB homology domain of Brf1. A model is proposed to explain how two distinct functional domains of Brf1 work in concert to dissociate TBP into monomers.