Transcription activation by FNR: evidence for a functional activating region 2

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• The FNR protein regulates gene transcription in Escherichia coli under anoxic conditions.
• FNR utilizes surface-exposed activating regions (AR1 and AR3) to interact with RNA polymerase (RNAP).
• A functional activating region 2 (AR2) in FNR was previously thought to be absent.

Purpose of the Study:

• To investigate the role of specific lysine residues (Lys49 and Lys50) in FNR function.
• To determine if these residues contribute to FNR's interaction with RNAP and transcriptional activation.
• To identify potential interactions between FNR and the RNAP alpha subunit.

Main Methods:

• Site-directed mutagenesis was used to substitute lysine residues (Lys49, Lys50) in FNR with alanine or glutamate.
• Transcriptional activity of FNR mutants was assessed at class I and class II FNR-dependent promoters.
• Oriented heterodimers were employed to analyze subunit-specific effects.
• Mutagenesis of the RNAP alpha subunit was performed to probe interactions.

Main Results:

• Substitution of Lys49 and Lys50 impaired transcription at class II promoters but not class I promoters.
• The impact of substitutions was greater with charged residues (glutamate) than uncharged residues (alanine).
• Only the downstream FNR subunit was affected at class II promoters.
• Evidence suggests Lys49 and Lys50 interact with a negatively charged region in the RNAP alpha subunit.

Conclusions:

• Lysine residues 49 and 50 of FNR constitute a functional activating region 2 (AR2).
• This AR2 is specifically involved in activating transcription at class II FNR-dependent promoters.
• The findings elucidate a novel mechanism of FNR-RNAP interaction and transcriptional control.