Promoter domain mediates guanosine tetraphosphate activation of the histidine operon | JoVE Visualize

Promoter domain mediates guanosine tetraphosphate activation of the histidine operon

Proceedings of the National Academy of Sciences of the United States of America|December 1, 1986

View abstract on PubMed

Summary

The alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp) significantly regulates the Salmonella typhimurium histidine operon. Mutations altering promoter sequences reduced ppGpp

Area of Science:

Molecular Biology
Microbial Genetics
Gene Regulation

Background:

The alarmone guanosine 5 -diphosphate 3 -diphosphate (ppGpp) is a key regulator of bacterial gene expression, particularly under stress conditions.
The histidine operon in Salmonella typhimurium is a well-studied system for understanding bacterial gene regulation.
Understanding ppGpp's role in operon regulation is crucial for deciphering bacterial stress responses.

Purpose of the Study:

To investigate the in vitro effects of ppGpp on the regulation of the Salmonella typhimurium histidine operon.
To identify specific DNA sequence elements involved in ppGpp-mediated transcriptional regulation.
To elucidate the mechanisms by which ppGpp activates or represses transcription.

Main Methods:

Analysis of wild-type and mutant Salmonella typhimurium histidine operon promoter expression using a DNA-dependent transcription-translation system.
Site-specific mutations were introduced using oligonucleotide-directed mutagenesis to alter the -10 hexamer region.
Deletion mutations were generated using BAL-31 nuclease to modify the region between the -10 hexamer and the transcription start site.

Main Results:

ppGpp strongly stimulated wild-type histidine operon expression (22-fold) in vitro with plasmid DNA templates.
Mutations enhancing the -10 hexamer homology to the consensus sequence significantly increased expression without ppGpp and reduced ppGpp stimulation.
A deletion mutation affecting the downstream region also modulated ppGpp regulation, similar to -10 hexamer mutations.

Conclusions:

Both the -10 hexamer sequence and the adjacent downstream region are critical for ppGpp-mediated regulation of the histidine operon.
The findings suggest a complex regulatory mechanism involving ppGpp that integrates promoter strength with regulatory signals.
Further studies are warranted to fully elucidate the molecular mechanisms of ppGpp's activation and repression roles in transcription.