Nucleic Acids Research Advance Access published online on August 16, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl559
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Elongation complexes of Thermus thermophilus RNA polymerase that possess distinct translocation conformations
1 Department of Cell Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine Stratford, NJ 08084, USA 2 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences 119991, Moscow, Russian Federation 3 APCG RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho Sayo Hyogo 679-5148 Japan 4 Lied Transplant Center Eppley Institute for Research in Cancer and Allied Diseases University of Nebraska Medical Center 10737A 986805 Nebraska Medical Center Omaha, Nebraska 68198 5 Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Schools of Medicine and Dentistry Birmingham, AL 35294, USA 6 Structural and Molecular Biology Laboratory, RIKEN Harima Institute at SPring-8 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan
*To whom correspondence should be addressed. Tel: 856 566 6274; Fax: 856 566 2881; Email: d.temiakov{at}umdnj.edu
Received May 22, 2006. Revised July 18, 2006. Accepted July 18, 2006.
We have characterized elongation complexes (ECs) of RNA polymerase from the extremely thermophilic bacterium, Thermus thermophilus. We found that complexes assembled on nucleic acid scaffolds are transcriptionally competent at high temperature (50–80°C) and, depending upon the organization of the scaffold, possess distinct translocation conformations. ECs assembled on scaffolds with a 9 bp RNA:DNA hybrid are highly stable, resistant to pyrophosphorolysis, and are in the posttranslocated state. ECs with an RNA:DNA hybrid longer or shorter than 9 bp appear to be in a pretranslocated state, as evidenced by their sensitivity to pyrophosphorolysis, GreA-induced cleavage, and exonuclease footprinting. Both pretranslocated (8 bp RNA:DNA hybrid) and posttranslocated (9 bp RNA:DNA hybrid) complexes were crystallized in distinct crystal forms, supporting the homogeneity of the conformational states in these complexes. Crystals of a posttranslocated complex were used to collect diffraction data at atomic resolution.
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