Amino-terminal amino acids modulate sigma-factor DNA-binding activity

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Prokaryotic transcription initiation factor sigma is crucial for RNA polymerase to recognize promoter DNA sequences.
• Previous genetic studies suggested sigma directly interacts with DNA at promoter regions (-10 and -35 consensus sequences).
• In vitro DNA binding studies with Escherichia coli sigma 70 revealed that only truncated forms, lacking amino-terminal amino acids, bind DNA.

Purpose of the Study:

• To investigate the function of the amino terminus of E. coli sigma 70 in regulating its DNA-binding capability.
• To determine if the amino terminus acts as an inhibitory domain for DNA binding.
• To examine if this inhibitory mechanism is conserved across different sigma factors.

Main Methods:

• Deletion analysis of the E. coli sigma 70 protein to identify inhibitory regions.
• In vitro DNA-binding assays using modified sigma 70 polypeptides.
• Trans-inhibition assays to assess the effect of the amino-terminal domain in isolation.
• Comparative analysis with alternative sigma factors (E. coli sigma 32 and Bacillus subtilis sigma K).

Main Results:

• Deletion analysis identified amino acids within the amino-terminal region 1.1 of sigma 70 as inhibitors of DNA binding.
• This amino-terminal inhibitory domain was shown to inhibit DNA binding by the carboxy-terminal DNA-binding domains, even in trans.
• Similar negative regulatory effects on DNA binding were observed for the amino-terminal extensions of E. coli sigma 32 and B. subtilis sigma K.

Conclusions:

• The amino terminus of sigma 70 acts as an intramolecular inhibitor of DNA binding.
• Transcription initiation is likely modulated by the structure and composition of the sigma-subunit's amino terminus.
• Sigma factors represent a class of intramolecularly regulated transcriptional effectors.