The MotA Transcriptional Activator of Bacteriophage T4 Binds to Its Specific DNA Site as a Monomer

Abstract

During bacteriophage T4 middle mode gene expression, the MotA transcription factor binds to T4 middle promoters at a -30 mot box consensus sequence to allow activation. Previous binding studies showed that MotA forms multiple gel-shifted complexes with DNA, and structural evidence suggested that MotA dimerizes upon DNA binding. We have shown that a short (13 bp) mot box DNA substrate binds MotA protein but fails to form slower migrating complexes. Therefore, the slower migrating complexes in gel shift assays are caused by DNA-mediated binding events. Competition experiments indicate that the slower migrating complexes are formed by nonspecific binding events, while the first-shifted complex is caused by specific binding to the mot box. Saturation binding experiments revealed that the stoichiometry of MotA binding to DNA is 1:1 in the first-shifted complex, while the slower complexes apparently contain MotA multimers. Gel shift assays using mixtures of MotA and a GST-MotA fusion protein supported the conclusion that the first-shifted complex contains one protein molecule bound to DNA. Furthermore, MotA monomers were cross-linked by glutaraldehyde under conditions where slower complexes exist, but not under conditions that lead to only the first-shifted complex. We conclude that MotA binds specifically to the mot box as a monomer and that additional nonspecific binding events require flanking DNA.

Architecture of the Bacteriophage T4 Activator MotA/Promoter DNA Interaction during Sigma Appropriation

“…This provided confidence that the FeBABE analyses were reporting the relevant position of MotA within the -appropriated complex. In addition, the positions of the cleavages were consistent with the binding of a monomer of MotA (rather than a dimer or other multimers) within the transcription complex; previous work has indicated that MotA alone binds as a monomer to the MotA box (45).…”

“…6B1). This has been observed previously when analyzing MotA binding by EMSA (46) or by fluorescence anisotropy (45). Although MotA binds to the MotA element as a monomer (45), EMSAs suggest that multimers of MotA associate with the protein-DNA complex as the concentration of MotA increases.…”

Refining the Binding of the Escherichia coli Flagellar Master Regulator, FlhD 4 C 2 , on a Base-Specific Level

“…Some transcriptional regulators bind their target sites as monomers (e.g., members of the AraC family and MotA from bacteriophage T4) (11,17), or as homomultimers (e.g., LacI, H-NS, and the LysR family) (4,6,34,52). Heteromeric transcriptional activators are unusual in bacteria but have been reported, such as the dimer of integration host factor, IHF-␣␤, the dimer of the regulator of capsular biosynthesis, RcsBA, and dimers of the nucleoprotein H-NS with another nucleoprotein, Hha or StpA (15,57).…”

Transcriptional takeover by σ appropriation: remodelling of the σ 70 subunit of Escherichia coli RNA polymerase by the bacteriophage T4 activator MotA and co-activator AsiA

“…This promoter binding in the presence of AsiA requires the DNA-binding region of the a subunits of RNA polymerase, suggesting that with a long enough incubation, a-DNA contacts can eventually compensate for the lack of contacts between s 70 region 4 and the 235 DNA element (Orsini et al, 2001). The T4 MotA protein is a transcription activator that binds as a monomer (Cicero et al, 1998;Li et al, 2002) to the MotA box element (Brody et al, 1983;Guild et al, 1988;Hinton, 1991;Schmidt & Kreuzer, 1992) with an apparent dissociation constant of 100-200 nM (Cicero et al, 1998;Sharma et al, 1999a). MotA also interacts with s 70 (Gerber & Hinton, 1996;Pande et al, 2002).…”