Effect of the non-conserved N-terminus on the DNA binding activity of the yeast TATA binding protein.

We have studied the DNA binding activity of recombinant yeast TATA Binding Protein (TBP) with particular interest in the role played by the non-conserved N-terminal domain. By comparing the DNA binding activity of wild type yeast TBP with a mutant form of TBP that lacks the non-conserved N-terminal domain (TBP delta 57), we have determined that the N-terminus of TBP alters both the shape and the stability of the TBP-DNA complex. Measurements of the DNA bending angle indicate that the N-terminus enhances the bending of the DNA that is induced by TBP binding and greatly destabilizes the TBP-DNA complex during native gel electrophoresis. In solution, the N-terminus has only a slight effect on the equilibrium dissociation constant and the dissociation rate constant. However, the N-terminal domain reduces the association rate constant in a temperature dependent manner and increases the apparent activation energy of the TBP-DNA complex formation by 3 kcal/mole. These data suggest that a conformational change involving the N-terminus of TBP may be one of the isomerization steps in the formation of a stable TBP-DNA complex.     

Signals for TBP/TATA box recognition

The TATA box-binding protein (TBP) recognizes its target sites (TATA boxes) by indirectly reading the DNA sequence through its conformation effects (indirect readout). Here, we explore the molecular mechanisms underlying indirect readout of TATA boxes by TBP by studying the binding of TBP to adenovirus major late promoter (AdMLP) sequence variants, including alterations inside as well as in the sequences flanking the TATA box. We measure here the dissociation kinetics of complexes of TBP with AdMLP targets and, by phase-sensitive assay, the intrinsic bending in the TATA box sequences as well as the bending of the same sequence induced by TBP binding. In these experiments we observe a correlation of the kinetic stability to sequence changes within the TATA recognition elements. Comparison of the kinetic data with structural properties of TATA boxes in known crystalline TBP/TATA box complexes reveals several "signals" for TATA box recognition, which are both on the single base-pair level, as well as larger DNA tracts within the TATA recognition element. The DNA bending induced by TBP on its binding sites is not correlated to the stability of TBP/TATA box complexes. Moreover, we observe a significant influence on the kinetic stability of alteration in the region flanking the TATA box. This effect is limited however to target sites with alternating TA sequences, whereas the AdMLP target, containing an A tract, is not influenced by these changes.     

The intercalating beta-hairpin of T7 RNA polymerase plays a role in promoter DNA melting and in stabilizing the melted DNA for efficient RNA synthesis

Phage T7 RNA polymerase contains within its single polypeptide all the elements for specific recognition and melting of its promoter DNA. Crystallographic studies indicate that a beta-hairpin (230-245) with an intercalating valine residue plays a role in promoter opening. We mutated V237 to several amino acids, deleted five amino acid residues at the tip of the hairpin, and mutated E242 and D240 at the base of the hairpin to define the roles of the tip and base of the hairpin in DNA strand separation. The affinity of the hairpin mutants for the promoter DNA was not significantly affected. Stopped-flow kinetic studies showed that the bimolecular rate of DNA binding and the observed rate of pre-initiation open complex formation that corresponds to the sum of DNA opening and closing steps were within 20 to 40 % of the wild-type polymerase. Yet, most mutants showed a smaller amount of the pre-initiation open complex at equilibrium, indicating that the individual rates of promoter opening and closing steps were altered in the mutants. The base mutants, E242A and D240A, showed both a lower rate of promoter opening and a higher rate of promoter closing, suggesting their role in stabilization of the open complex. The V237D and the deletion mutant showed mainly a lower rate of promoter opening, suggesting that the tip of the hairpin may nucleate DNA opening. The defect in pre-initiation open complex formation affected downstream steps such as the rate of the first phosphodiester bond formation step, but did not affect significantly the apparent K(d) of initiating GTPs. We propose that D240 and E242 anchor the hairpin to the DNA and position the tip of the hairpin to allow V237 to intercalate and distort the DNA during open complex formation. The interactions of E242 and D240 with the upstream junction of the melted dsDNA promoter also align the template strand within the active site for efficient RNA synthesis.     

Visualizing induced fit in early assembly of the human signal recognition particle

Assembly of almost all ribonucleoprotein complexes involves induced fit in the RNA and, thus, formation of one or more intermediate states. In assembly of the human signal recognition particle (SRP), we show that SRP19 binding to SRP RNA involves obligatory intermediates. An apparent discrepancy exists between the ratio of dissociation and association rate constants, determined in a partitioning experiment, and the equilibrium binding constant; this kinetic signature reflects formation of a stable intermediate in assembly of the ribonucleoprotein complex. Assembly intermediates were observed directly by time-resolved footprinting. SRP19 binds rapidly to SRP RNA to form an initial labile, but structurally specific, encounter complex involving both helices III and IV. Two subsequent steps of structural consolidation yield the native RNA-protein interface. SRP19 binding stabilizes helix IV in the region recognized by SRP54, consistent with protein-protein cooperativity mediated in part by mutual recognition of similar RNA structures. This mechanism illustrates principles general to ribonucleoprotein assembly reactions that rely on recruitment of architectural RNA binding proteins.     

Two novel Drosophila TAF(II)s have homology with human TAF(II)30 and are differentially regulated during development

TFIID is a multiprotein complex composed of the TATA binding protein (TBP) and TBP-associated factors (TAF(II)s). The binding of TFIID to the promoter is the first step of RNA polymerase II preinitiation complex assembly on protein-coding genes. Yeast (y) and human (h) TFIID complexes contain 10 to 13 TAF(II)s. Biochemical studies suggested that the Drosophila (d) TFIID complexes contain only eight TAF(II)s, leaving a number of yeast and human TAF(II)s (e.g., hTAF(II)55, hTAF(II)30, and hTAF(II)18) without known Drosophila homologues. We demonstrate that Drosophila has not one but two hTAF(II)30 homologues, dTAF(II)16 and dTAF(II)24, which are encoded by two adjacent genes. These two genes are localized in a head-to-head orientation, and their 5' extremities overlap. We show that these novel dTAF(II)s are expressed and that they are both associated with TBP and other bona fide dTAF(II)s in dTFIID complexes. dTAF(II)24, but not dTAF(II)16, was also found to be associated with the histone acetyltransferase (HAT) dGCN5. Thus, dTAF(II)16 and dTAF(II)24 are functional homologues of hTAF(II)30, and this is the first demonstration that a TAF(II)-GCN5-HAT complex exists in Drosophila. The two dTAF(II)s are differentially expressed during embryogenesis and can be detected in both nuclei and cytoplasm of the cells. These results together indicate that dTAF(II)16 and dTAF(II)24 may have similar but not identical functions.