Analysis of the sequence-specific interactions between Tus, the replication arrest protein of Escherichia coli, and its DNA binding sequence

• Main Author: Coskun-Ari, Fatma Filiz
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-1996
• Subjects: Molecular biology
• ISBN: 0591054019; 9780591054019

Regulation of many biological events is mediated by specific protein-DNA interactions. Since there is no simple code for protein-DNA recognition, one of the major questions of present day molecular biology is how proteins interact with their target DNA binding sites. Termination of DNA replication in E. coli is mediated by the specific interactions between the Tus protein and the terminator (Ter) sites on the chromosome. Binding of Tus to the Ter sites causes formation of asymmetric protein-DNA complexes that arrest replication forks in an orientation-dependent fashion. The objective of this study is to determine the critical base pairs and their contribution to the protein-DNA interactions in terms of Tus-Ter binding and in vivo functioning of the protein-DNA complex. The binding affinities of purified Tus protein to the mutant Ter sites carrying single-base pair substitutions at 16 positions were quantitatively measured using the equilibrium competition binding assay. The replication arrest abilities of mutant Ter sites were also examined using a Y-fork assay to determine the effect of each base pair substitution in vivo. The results from in vitro binding experiments demonstrated that all three possible base pair substitutions made at positions 8-19 had large effects (higher than 1.5 kcal/mol) on the stability of the Tus-Ter complex. In agreement with this observation, the mutants of these positions also showed significantly low or no replication arrest activities in vivo, confirming the fact that interactions between Tus and these nucleotides in the major and minor grooves of DNA were crucial for the complex formation and Tus activity. Substitutions introduced at positions 6, 20 and 21 had a varied free energy change response, suggesting that these nucleotides contribute to, but not absolutely critical for, Tus-Ter binding in vitro. However, the data from in vivo experiments revealed that the Ter mutants of these positions were not as efficient as the WT Tus-TerB complex in arresting replication forks. This observed requirement of wild-type nucleotides at positions 6, 20 and 21 suggested that Tus might be extending within the DNA major groove across from one end to the other. Therefore, it is possible that substitution of the base pairs located at the edges of the Ter sequence might cause conformational changes in Tus that result in reduced replication arrest activity. On the other hand, results from both in vitro and in vivo experiments agreed that the residue of position 7 was the only dispensable base pair for Tus-Ter binding and in vivo functioning of the complex. The data obtained from our mutational analysis, thus, indicate that 15 out of 16 nucleotides of the Ter sequence are important for the Tus-Ter interactions and necessary for biological replication arrest function of the protein-DNA complex. Finally, three new potential Ter sites designated TerG, TerH and TerI were identified by searching the Genbank database for putative Ter sites on the bases of sequence similarity. Having the information on the effect of individual base pairs in Tus-Ter complex, we were able to predict the binding affinity of Tus to these putative Ter sites and their replication arrest efficiencies in vivo. Therefore, once the sequence of entire E. coli genome is available, all potential and pseudo Ter sites can then easily be identified.