Abstract
23 amino acid substitutions were made in the C7 and C3 regions of pspFDeltaHTH, a protein required to convert sigma(54) closed promoter complexes to open complexes. These mutants were assayed for transcriptional competence, for the ability to hydrolyze ATP, for their multimerization state, and for their ability to interact with sigma(54) and its holoenzyme. C7 region mutants caused the protein to assume a compact form. This property could be mimicked by the addition of ATP, implying that compaction via C7 and ATP is part of the activation process. A number of C3 mutants were important for energy coupling, as indicated previously for several members of this activator family (, ). However, a patch within C3 influenced oligomerization. The C3 region was especially important in interacting with sigma(54) during the transition state but not important in inducing sigma(54) holoenzyme to engage the nontemplate strand of the promoter. It is proposed that both regions contain deterrent functions that prevent premature activation. Overall, the results imply unexpected roles for the C7 and C3 regions of this protein family during promoter activation.
Highlights
Transcription of genes with promoters recognized by 54 holoenzyme requires enhancer-binding activator proteins (3)
The C3 region was especially important in interacting with 54 during the transition state but not important in inducing 54 holoenzyme to engage the nontemplate strand of the promoter
The central domain is required for activation, since it contains the information that allows the coupling of ATP hydrolysis to the melting of the DNA by holoenzyme
Summary
Plasmids and Proteins—The plasmid pMJ15 contains His6pspF⌬HTH (18). The QuikChange mutagenesis kit (Stratagene) was used for site-directed mutagenesis. 75 nM 54 RNA polymerase was incubated with 0.5 mM GTP, 0.5 mM ATP, and 0.5 mM UTP in 1ϫ buffer B (50 mM Tris-HCl at pH 7.5, 50 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 2 mM -mercaptoethanol) (24) for 20 min at 37 °C before 1 l of CTP mixture (50 M CTP and 0.2 Ci/l [␣-32P]CTP) was added. 5 M pspF⌬HTH and its mutants were incubated in 1ϫ buffer A (25 mM Hepes at pH 7.5, 20 mM MgCl2, 10 mM KCl, 2 mM -mercaptoethanol) before ATP␥S1 mix (0.6 mM ATP␥S, 69 M [␥-35S]ATP) was added to the 20-l reaction. This was incubated at 37 °C for 4 min.
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