Structural basis for substrate loading in bacterial RNA polymerase

• Published in: Nature 2007-07, Vol.448 (7150), p.163-168
• Main Authors: Vassylyev, Dmitry G., Vassylyeva, Marina N., Zhang, Jinwei, Palangat, Murali, Artsimovitch, Irina, Landick, Robert
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Aminoglycosides - pharmacology; Antibiotics; Bacteria; Bacterial Proteins - metabolism; Biochemistry; Biological and medical sciences; Catalysis; Crystalline structure; Crystallization; Crystallography, X-Ray; Displacement; DNA-Directed RNA Polymerases - antagonists & inhibitors; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - metabolism; Elongation; Freezing; Fundamental and applied biological sciences. Psychology; Gene expression; Genetic transcription; Genetics; Humanities and Social Sciences; Inhibitors; Insertion; Models, Molecular; Molecular biophysics; multidisciplinary; Multiprotein Complexes - chemistry; Multiprotein Complexes - metabolism; Nucleotides - metabolism; Polymerase; Protein Conformation; Protein Folding; Protein Structure, Secondary; Ribonucleic acids; RNA polymerase; RNA polymerases; Science; Science (multidisciplinary); Structure in molecular biology; Substrate Specificity; Substrates; Thermus thermophilus; Thermus thermophilus - enzymology; Transcription, Genetic
• ISSN: 0028-0836; 1476-4687; 1476-4679
• DOI: 10.1038/nature05931

The mechanism of substrate loading in multisubunit RNA polymerase is crucial for understanding the general principles of transcription yet remains hotly debated. Here we report the 3.0-Å resolution structures of the Thermus thermophilus elongation complex (EC) with a non-hydrolysable substrate analogue, adenosine-5′-[(α,β)-methyleno]-triphosphate (AMPcPP), and with AMPcPP plus the inhibitor streptolydigin. In the EC/AMPcPP structure, the substrate binds to the active (‘insertion’) site closed through refolding of the trigger loop (TL) into two α-helices. In contrast, the EC/AMPcPP/streptolydigin structure reveals an inactive (‘preinsertion’) substrate configuration stabilized by streptolydigin-induced displacement of the TL. Our structural and biochemical data suggest that refolding of the TL is vital for catalysis and have three main implications. First, despite differences in the details, the two-step preinsertion/insertion mechanism of substrate loading may be universal for all RNA polymerases. Second, freezing of the preinsertion state is an attractive target for the design of novel antibiotics. Last, the TL emerges as a prominent target whose refolding can be modulated by regulatory factors. RNA polymerase up close Two complementary papers this week focus on the structure and function of bacterial RNA polymerase. In the first, the enzyme is bound to the DNA template and RNA product, to give a close-up of the transcription elongation complex. The structure reveals details of the DNA-to-RNA transcription process, vital to all living cells. In the second paper, the RNA polymerase elongation complex is pictured bound to various substrate analogues and to an antibiotic, revealing the mechanism of substrate loading and antibiotic inhibition. Comparisons between the structures of human and bacteria RNA polymerase should aid in drug design: RNA polymerase is a target of antibiotics, including rifamycin and its derivatives. Crystal structures of bacterial RNA polymerase elongation complexes bound to NTP substrate analogues with an antibiotic, revealing the mechanism of substrate loading and antibiotic inhibition.