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Discrimination against deoxyribonucleotide substrates by bacterial RNA polymerase
Vladimir Svetlov1, Dmitry G Vassylyev, Irina Artsimovitch
1Department of Microbiology, The Ohio State University, Columbus, Ohio 43210, USA.
The Journal of Biological Chemistry
|July 21, 2004
Summary
Bacterial RNA polymerases distinguish correct nucleotide substrates using a conserved residue in the active site, unlike single-subunit polymerases. This finding reveals key mechanisms for accurate RNA synthesis in bacteria.
Area of Science:
- Molecular Biology
- Biochemistry
- Enzymology
Background:
- Nucleic acid polymerases prevent non-cognate substrate incorporation based on base and sugar.
- Substrate selection mechanisms are known for single-subunit polymerases but not multisubunit RNAPs.
Purpose of the Study:
- To identify determinants of substrate binding in multisubunit bacterial RNA polymerases (RNAPs).
- To investigate the role of the beta' subunit Asn residue in substrate selection.
Main Methods:
- Molecular modeling of Thermus thermophilus RNAP-NTP complex.
- Site-directed mutagenesis of Escherichia coli RNAP beta' subunit Asn(458).
- In vitro functional assays to assess substrate discrimination and RNA chain extension.
Main Results:
- Molecular modeling identified a conserved beta' subunit Asn residue potentially involved in ribose selection.
- Mutations in E. coli RNAP beta' Asn(458) abolished discrimination between ribo- and deoxyribonucleotide substrates.
- Mutations also led to defects in RNA chain extension.
Conclusions:
- Bacterial RNAPs likely recognize the sugar moiety within the active (insertion) site.
- This contrasts with single-subunit RNAPs that select substrates in a pre-insertion site.
- A conserved beta' subunit Asn residue is crucial for sugar moiety recognition in bacterial RNAPs.