X-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides

• Published in: Proceedings of the National Academy of Sciences - PNAS 2011-03, Vol.108 (9), p.3566-3571
• Main Authors: Gleghorn, Michael L, Davydova, Elena K, Basu, Ritwika, Rothman-Denes, Lucia B, Murakami, Katsuhiko S
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; Active sites; Amino Acids - metabolism; Bacteriophage N4 - enzymology; BACTERIOPHAGES; BASIC BIOLOGICAL SCIENCES; Binding Sites; Biocatalysis; Biological Sciences; Cadmium; CATALYSIS; Catalytic Domain; Chromium; CONFORMATIONAL CHANGES; Coordination polymers; CRYSTAL STRUCTURE; Crystallography, X-Ray; Crystals; DNA; DNA POLYMERASES; DNA, Viral - chemistry; DNA, Viral - genetics; DNA-directed RNA polymerase; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - metabolism; ELONGATION; Enzymes; Guanosine Diphosphate - metabolism; Guanosine Triphosphate - metabolism; Initiation complex; Ionizing radiation; Metals; Metals - metabolism; Models, Molecular; Molecules; NUCLEIC ACIDS; NUCLEOTIDES; Nucleotides - chemistry; Nucleotides - metabolism; Phages; POLYMERASES; Protein Conformation; Ribonucleic acid; RNA; RNA phages; RNA polymerase; RNA POLYMERASES; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA, Messenger - metabolism; Substrate Specificity; SUBSTRATES; TRANSCRIPTION; Transcription, Genetic; TRANSFER REACTIONS; Virion - enzymology; X-rays
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1016691108

We have determined the X-ray crystal structures of the pre- and postcatalytic forms of the initiation complex of bacteriophage N4 RNA polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit RNA polymerase. As observed during T7 RNA polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the O helix, but only the correct base pairing between the +2 substrate and DNA base is able to complete the O-helix conformational transition. Substrate binding also facilitates catalytic metal binding that leads to alignment of the reactive groups of substrates for the nucleotidyl transfer reaction. Although all nucleic acid polymerases use two divalent metals for catalysis, they differ in the requirements and the timing of binding of each metal. In the case of bacteriophage RNA polymerase, we propose that catalytic metal binding is the last step before the nucleotidyl transfer reaction.