Related Experiment Videos
Distinct DNA elements contribute to Rap1p affinity for its binding sites.
Valerio Del Vescovo1, Veronica De Sanctis, Alessandro Bianchi
1Dipartimento di Biologia Cellulare e dello Sviluppo, Università La Sapienza di Roma, Rome, Italy.
Journal of Molecular Biology
|April 28, 2004
Summary
Researchers mapped the DNA-binding site of Saccharomyces cerevisiae Rap1 protein. Findings reveal Rap1 interacts with DNA beyond consensus sequences, explaining its promoter binding flexibility.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The Saccharomyces cerevisiae regulatory protein Rap1 is crucial for gene expression.
- Rap1 possesses two tandem Myb-like DNA-binding sub-domains that interact with DNA hemisites separated by a linker sequence.
Purpose of the Study:
- To precisely map the thermodynamically defined DNA-binding site of the Rap1 protein.
- To investigate Rap1's interaction with DNA sequences beyond established consensus sites.
Main Methods:
- Primer extension method coupled with electrophoretic separation of bound and unbound DNA.
- Analysis of Rap1 binding to modified and minimal DNA sequences.
Main Results:
- Rap1 binding extends 3 base pairs beyond the 5' end of the published consensus sequence.
- This extended interaction occurs where the DNA minor groove faces the protein, potentially explaining Rap1-induced DNA bending.
- Minimal DNA-binding sites, including a single hemisite with a spacer, bind Rap1 with lower affinity.
Conclusions:
- The study refines the understanding of the Rap1 DNA-binding site, revealing interactions beyond canonical sequences.
- These extended interactions and binding to minimal sites may explain Rap1's in vivo binding at promoters lacking canonical sites.
- Findings contribute to understanding gene regulation by Rap1 in Saccharomyces cerevisiae.