Plant expression signals of the Agrobacterium T-cyt gene.

Within the 5' and 3' non-coding regions of the T-cyt gene from the octopine T-DNA of Agrobacterium tumefaciens sequences required for expression of this gene in plant cells were identified by deletion mutagenesis. The results show that 184 bp of the 5' non-coding region and 270 bp of the 3' non-coding region are sufficient for wild-type expression. Within the 5' non-coding region two essential expression signals were identified: (1.) an activator element located between -185 and -129 with respect to the ATG start codon and (2.) one out of two TATA boxes. Deletions of the activator element or the two TATA boxes resulted in nonfunctional genes. Deletion of the upstream TATA box and both putative CAAT boxes did not significantly affect expression. Within the 3' non-coding region, the polyadenylation box most distal to the stop codon was not essential for expression, but sequences more upstream, including a second polyadenylation box were found to be required for wild-type expression.     

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.     

Selective binding of the TATA box-binding protein to the TATA box-containing promoter: analysis of structural and energetic factors.

We report the results of an energy-based exploration of the components of selective recognition of the TATA box-binding protein (TBP) to a TATA box sequence that includes 1) the interaction between the hydrophobic Leu, Pro, and Phe residues of TBP with the TA, AT, AA, TT, and CG steps, by ab initio quantum mechanical calculations; and 2) the free energy penalty, calculated from molecular dynamics/potential of mean force simulations, for the conformational transition from A-DNA and B-DNA into the TA-DNA form of DNA observed in a complex with TBP. The GTAT, GATT, GAAT, and GTTT tetramers were explored. The results show that 1) the discrimination of TA, AT, AA, TT, or CG steps by TBP cannot rest on their interaction with the inserting Phe side chains; 2) the steric clash between the bulky and hydrophobic Pro and Leu residues and the protruding -NH2 group of guanine is responsible for the observed selectivity against any Gua-containing basepair; 3) the Pro and Leu residues cannot selectively discriminate among TA, AT, AA, or TT steps; and 4) the calculated energy required to achieve the TA-DNA conformation of DNA that is observed in the complex with TBP appears to be a key determinant for the observed selectivity against the AT, AA, and TT steps. The simulations also indicate that only the TA step can form a very efficient interbase hydrogen bond network in the TA-DNA conformation. Such an energetically stabilizing network is not achievable in the AA and TT steps. While it is viable in the AT step, structural constraints render the hydrogen bonding network energetically ineffective there.