Background: Cellular RNA polymerases start transcription by de novo RNA priming. Results: Structures and biochemical studies of initially transcribing complexes elucidate the de novo transcription initiation and early stage of RNA transcription. Conclusion: 5Ј-end of RNA in the transcribing complex starts ejection from core enzyme. Significance: Insights from this study can be applicable to all cellular RNA polymerases.

Telomerase is an RNA-protein complex (RNP) that extends telomeric DNA at the 3' ends of chromosomes using its telomerase reverse transcriptase (TERT) and integral template-containing telomerase RNA (TER). Its activity is a critical determinant of human health, affecting aging, cancer, and stem cell renewal. Lack of atomic models of telomerase, particularly one with DNA bound, has limited our mechanistic understanding of telomeric DNA repeat synthesis. We report the 4.8 Å resolution cryoelectron microscopy structure of active Tetrahymena telomerase bound to telomeric DNA. The catalytic core is an intricately interlocked structure of TERT and TER, including a previously structurally uncharacterized TERT domain that interacts with the TEN domain to physically enclose TER and regulate activity. This complete structure of a telomerase catalytic core and its interactions with telomeric DNA from the template to telomere-interacting p50-TEB complex provides unanticipated insights into telomerase assembly and catalytic cycle and a new paradigm for a reverse transcriptase RNP.

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 þ2 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. D NA-dependent RNA polymerases (RNAPs) transcribe DNA genetic information into RNA and play a central role in gene expression. RNAP catalyzes a nucleotidyl transfer reaction, which is initiated by the nucleophilic attack of an O3′ oxyanion at the RNA 3′ terminus to the α-phosphate (αP) of the incoming nucleotide, resulting in phosphodiester bond formation and release of pyrophosphate (PPi). Both single-subunit T7 phage-like RNAPs and the multisubunit cellular RNAPs possess two nucleotide-binding sites for loading the RNA 3′ end (P site) and the incoming NTP (N site) (1, 2). A two metal-ion catalytic mechanism has been proposed, as the enzyme possesses two divalent catalytic and nucleotide-binding metal cations chelated by two or three conserved Asp residues (3). The catalytic metal is a Lewis acid, coordinating the RNA 3′-OH lowering its pK a and facilitating the formation of the attacking oxyanion. The nucleotide-binding metal is coordinated by the triphosphate of the incoming nucleotide and stabilizes a pentacovalent phosphate intermediate during the reaction. Both metal ions are proposed to have octahedral coordination at physiological Mg 2þ concentrations (4). During transcript elongation, RNAP carries out the loading of a single nucleotide substrate at the N site followed by a nucleotidyl transfer reaction with the RNA 3′ end at the P site; this cycle is repeated as elongation proceeds. X-ray crystal structures of the single-subunit T7 phage RNAP (2, 5) have depicted the process of transcript elongation in detail and reveal a conformational change of the Fingers subdomain during substrate loading to the active site as also observed in the A family of DNA polymerases (DNAPs) (6, 7).Initiation is the only step in the entire transcription process where two nucleotide substrates are loaded at the active site followed by a nucleotidyl transfer reaction. Compared with elongation, the process of initiation has not been well characterized by X-ray crystallography. An X-ray crystal structure of T7 RNAP initiation complex...

Background:The two-metal-ion mechanism of the nucleotidyl transfer reaction by RNA/DNA polymerases has not been adequately elucidated due to lack of temporal resolution. Results: Soak-trigger-freeze x-ray crystallography revealed structures of natural, time-resolved intermediates of transcript initiation. Conclusion: First structural evidence shows that catalytic metal binds after the nucleotide and nucleotide-binding metal and right before reaction chemistry. Significance: Time-dependent soak-trigger-freeze x-ray crystallography can yield functionally relevant high resolution information to study enzyme reactions.

The nucleotidyl transfer reaction leading to formation of the first phosphodiester bond has been followed in real-time by Raman microscopy, as it proceeds in single crystals of the N4 phage virion RNA polymerase (RNAP). The reaction is initiated by soaking NTP substrates and divalent cations into the RNAP and promoter DNA complex crystal, where the phosphodiester bond formation is completed in about 40 minutes. This slow reaction allowed us to monitor the changes of RNAP and DNA conformations as well as bindings of substrate and metal through Raman spectra taken every 5 minutes. Recently published snapshot X-ray crystal structures along the same reaction pathway assisted the spectroscopic assignments of changes in the enzyme and DNA, while isotopically labeled NTP substrates allowed differentiation of the Raman spectra of bases in substrates and DNA. We observed that substrates are bound at 2-7 minutes after commencing soaking, the O-helix completes its conformational change, and that binding of both divalent metals required for catalysis in the active site changes the conformation of the ribose triphosphate at position +1. These are followed by a slower decrease of NTP triphosphate groups due to phosphodiester bond formation that reaches completion at about 15 minutes, and even slower complete release of the divalent metals at about 40 minutes. We have also shown that the O-helix movement can be driven by substrate binding only. The kinetics of the in crystallo nucleotidyl transfer reaction revealed in this study suggest that soaking the substrate and metal into the RNAP-DNA complex crystal for a few minutes generates novel and uncharacterized intermediates for future X-ray and spectroscopic analysis.