Interaction of the Escherichia coli Replication Terminator Protein (Tus) with DNA:  A Model Derived from DNA-Binding Studies of Mutant Proteins by Surface Plasmon Resonance

Abstract

The Escherichia coli replication terminator protein (Tus) binds tightly and specifically to termination sites such as TerB in order to halt DNA replication. To better understand the process of Tus-TerB interaction, an assay based on surface plasmon resonance was developed to allow the determination of the equilibrium dissociation constant of the complex (K D ) and association and dissocation rate constants for the interaction between Tus and various DNA sequences, including TerB, single-stranded DNA, and two nonspecific sequences that had no relationship to TerB. The effects of factors such as the KCl concentration, the orientation and length of the DNA, and the presence of a single-stranded tail on the binding were also examined. The K D measured for the binding of wild type and His 6 -Tus to TerB was 0.5 nM in 250 mM KCl. Four variants of Tus containing single-residue mutations were assayed for binding to TerB and the nonspecific sequences. Three of these substitutions (K89A, R198A, and Q250A) increased K D by 200-300-fold, whereas the A173T substitution increased K D by 4000-fold. Only the R198A substitution had a significant effect on binding to the nonspecific sequences. The kinetic and thermodynamic data suggest a model for Tus binding to TerB which involves an ordered series of events that include structural changes in the protein.

The Escherichia coli Tus–Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

“…The 94-bp 3xTerB 'restrictive' module, as arranged on the Watson (5 0 -3 0 ) strand in the yeast genome, consisted of the following sequence: 5 0 -ATAAGTATGTTGTAACTAAAGTTAAGCCTGTATTCGAATAAGTATGT TGTAACTAAAGTTGACAGATCTTCAAATAAGTATGTTGTAACTAAAG-3 0 . The 21-bp TerB-restrictive sequences (that have been previously validated by surface plasmid resonance 18 ) are indicated in bold font. The 94-bp 3xTerB 'permissive' module used in this study was antiparallel to the 3xTerB 'restrictive' module.…”

“…TerB sequence was chosen for these analyses because the TusTerB interaction is well characterized 18 , and the TerB sequence has the strongest RF-pausing efficiency in E. coli 19 . We inserted three TerB sequences interspersed with two short spacer sequences adjacent to two well-characterized and early firing origins, ARS305 on chromosome III (ChrIII) and ARS607 on chromosome VI (ChrVI) (Fig.…”

Homomeric Ring Assemblies of Eukaryotic Sm Proteins Have Affinity for Both RNA and DNA

“…Primers were designed to incorporate NdeI and EcoRI restriction sites at the 5Ј and 3Ј ends of the genes, respectively. Genes were inserted into the expression plasmid pETMCSIII, which relies on transcription by T7 RNA polymerase and results in a hexa-His fusion at the N terminus of expressed proteins (26). The plasmid was transformed into the BL21(DE3)/pLysS Escherichia coli strain for expression.…”

Transition state for protein–DNA recognition

“…Some proteins diffuse mainly in 1D over long distances (2), whereas others favor a 3D pathway (3). Once at the target site, proteins without an enzymatic activity may bind to DNA along a single kinetic route (4)(5)(6)(7)(8)(9) or parallel routes (10,11). Each route may either be two-state (4-6, 10) or include populated kinetic intermediates (7)(8)(9)(10)(11).…”

“…Binding experiments that compare the kinetics of recognition of cognate and noncognate sequences and of wild-type sites and point mutants showed that some proteins do not display any level of sequence discrimination in the transition state for binding (4,5,17,18). Other proteins do discriminate between sequences in the transition state (6,19,20), sometimes as much as in the final complex (19,20). The detailed energetics of sequence readout and unspecific protein-DNA backbone interactions may also be analyzed by the effect that mutations exert on the binding kinetics (5,6,20,21).…”