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Conformational changes of the upstream DNA mediated by H-NS and FIS regulate E. coli RrnB P1 promoter activity
H Afflerbach1, O Schröder, R Wagner
1Institut für Physikalische Biologie, Heinrich-Heine-Universität D usseldorf, Universitätsstr. 1, Düsseldorf, D-40225, Germany.
Abstract:
The two proteins FIS and H-NS had previously been shown to regulate ribosomal RNA (rRNA) transcription by interacting with the promoter upstream DNA. FIS is known as an activator whereas H-NS had been demonstrated to function as a repressor. Details of the antagonistic control mechanisms are not yet solved. Here, we have addressed the question how the two proteins cooperate to exert both, positive and negative control of rRNA transcription. By mobility shift experiments and footprinting studies we show that FIS and H-NS binding sites partially overlap but appear to interact with different sites of a curved DNA helix. Although not mutually exclusive, the two proteins compete each other for binding. Both proteins, by changing the DNA curvature, effect circularization reactions of DNA fragments in different ways. Our results imply that binding of the proteins induces alternate DNA conformations with favourable or unfavourable topology for the formation of active transcription complexes. Together the findings presented here help to answer some of the open questions about the concerted molecular mechanism of transcription factors for the regulation of stable RNA synthesis.
Insights
FIS and H-NS proteins cooperatively regulate ribosomal RNA (rRNA) transcription by altering DNA curvature and competing for binding sites. This mechanism explains how these factors achieve both activation and repression of rRNA synthesis.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Ribosomal RNA (rRNA) transcription is crucial for cell growth and is regulated by proteins.
- FIS protein acts as an activator, while H-NS protein functions as a repressor of rRNA transcription.
- The precise antagonistic mechanisms of FIS and H-NS remain incompletely understood.
Purpose of the Study:
- To elucidate the cooperative mechanisms by which FIS and H-NS regulate rRNA transcription.
- To investigate how these two proteins achieve both positive and negative control over rRNA synthesis.
Main Methods:
- Electrophoretic mobility shift assays (EMSA) were employed to study protein-DNA interactions.
- DNA footprinting studies were conducted to map protein binding sites.
- DNA fragment circularization assays were used to assess DNA conformational changes.
Main Results:
- FIS and H-NS binding sites on promoter upstream DNA partially overlap.
- These proteins compete for binding, indicating an antagonistic interaction.
- Both proteins modulate DNA curvature, influencing DNA topology and transcription complex formation.
Conclusions:
- FIS and H-NS binding induces distinct DNA conformations, affecting transcription initiation.
- The interplay between FIS and H-NS provides a molecular basis for the dual regulation of stable RNA synthesis.
- These findings contribute to understanding the concerted action of transcription factors in gene regulation.