Bridge helix and trigger loop perturbations generate superactive RNA polymerases
| Autor: | Robert O. J. Weinzierl, Simone C. Wiesler, Dominika Trzaska, Hannah C. Carney, Lin Tan |
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| Jazyk: | angličtina |
| Rok vydání: | 2008 |
| Předmět: |
Models
Molecular Protein Conformation Protein subunit Molecular Sequence Data RNA Archaeal General Biochemistry Genetics and Molecular Biology 03 medical and health sciences chemistry.chemical_compound Protein structure RNA polymerase Amino Acid Sequence lcsh:QH301-705.5 Polymerase 030304 developmental biology 0303 health sciences biology Agricultural and Biological Sciences(all) Biochemistry Genetics and Molecular Biology(all) 030302 biochemistry & molecular biology Methanocaldococcus jannaschii RNA DNA-Directed RNA Polymerases biology.organism_classification Archaea Biochemistry chemistry lcsh:Biology (General) Helix biology.protein Biophysics Nucleic acid Mutagenesis Site-Directed General Agricultural and Biological Sciences Research Article |
| Zdroj: | Journal of Biology, Vol 7, Iss 10, p 40 (2008) Journal of Biology |
| Popis: | Background Cellular RNA polymerases are highly conserved enzymes that undergo complex conformational changes to coordinate the processing of nucleic acid substrates through the active site. Two domains in particular, the bridge helix and the trigger loop, play a key role in this mechanism by adopting different conformations at various stages of the nucleotide addition cycle. The functional relevance of these structural changes has been difficult to assess from the relatively small number of static crystal structures currently available. Results Using a novel robotic approach we characterized the functional properties of 367 site-directed mutants of the Methanocaldococcus jannaschii RNA polymerase A' subunit, revealing a wide spectrum of in vitro phenotypes. We show that a surprisingly large number of single amino acid substitutions in the bridge helix, including a kink-inducing proline substitution, increase the specific activity of RNA polymerase. Other 'superactivating' substitutions are located in the adjacent base helices of the trigger loop. Conclusion The results support the hypothesis that the nucleotide addition cycle involves a kinked bridge helix conformation. The active center of RNA polymerase seems to be constrained by a network of functional interactions between the bridge helix and trigger loop that controls fundamental parameters of RNA synthesis. |
| Databáze: | OpenAIRE |
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