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Active center rearrangement in RNA polymerase initiation complex
A Mustaev1, M Kashlev, E Zaychikov
1Public Health Research Institute, New York, New York 10016.
The Journal of Biological Chemistry
|September 15, 1993
Summary
Histidine 1237 in Escherichia coli RNA polymerase active center binds RNA primers up to 9 nucleotides. This finding suggests RNA polymerase active site filling precedes promoter escape, linking catalysis to translocation.
Area of Science:
- Molecular Biology
- Enzymology
- Biochemistry
Background:
- The beta subunit of Escherichia coli RNA polymerase contains His1237, located at the active center's 5' face.
- This residue is critical for interacting with the priming substrate's gamma-phosphate.
Purpose of the Study:
- To investigate the role of His1237 in RNA synthesis and its spatial relationship within the RNA polymerase active center.
- To explore the implications of active center occupancy on enzyme function and promoter escape.
Main Methods:
- Utilizing cross-linking techniques to covalently attach nucleotides to His1237.
- Synthesizing RNA chains using His1237-cross-linked nucleotides as primers.
- Analyzing the capacity of the RNA polymerase active center to accommodate RNA chains.
Main Results:
- His1237 can be cross-linked to the gamma-phosphate of the priming nucleotide.
- RNA chains up to 9 nucleotides in length can be synthesized using His1237-bound primers.
- A significant RNA mass can be held within the active center, positioned between His1237 and the catalytic site.
Conclusions:
- The active center of RNA polymerase can accommodate substantial RNA lengths before promoter clearance.
- Active site "filling" with RNA may be coupled to promoter escape and saltatory translocation.
- His1237 plays a key role in positioning the primer and potentially regulating RNA polymerase translocation.