4ff4

N4 mini-vRNAP transcription initiation complex, 4 min after soaking GTP, ATP and Mn

spinqualitylabelsall models 4ff4, resolution 2.03Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 4ff4 is a 4 chain structure with sequence from Escherichia virus N4. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.026Å Ligands: 5GP, AMP, MN, PO4, POP Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function RPOLV_BPN4 DNA-dependent RNA polymerase, which is injected into the host upon infection and transcribes the phage early genes from promoters that have a 5-bp stem-3 nt loop hairpin structure. Publication Abstract from PubMed The challenge for structural biology is to understand atomic-level macromolecular motions during enzymatic reaction (1-3). X-ray crystallography can reveal high-resolution structures; however, one perceived limitation is that it reveals only static views. Here we use time-dependent soak-trigger-freeze X-ray crystallography, namely, soaking nucleotide and divalent metal into the bacteriophage RNA polymerase (RNAP)-promoter DNA complex crystals to trigger the nucleotidyl transfer reaction and freezing crystals at different time points, to capture real-time intermediates in the pathway of transcription. In each crystal structure, different intensities and shapes of electron density maps corresponding to the nucleotide and metal were revealed at the RNAP active site that allow watching the nucleotide and metal bindings and the phosphodiester bond formation in a time perspective. Our study provides temporal order of substrate assembly and metal co-factor binding at the active site of enzyme that completes our understanding of the two-metal-ion mechanism (4-6) and fidelity mechanism in single-subunit RNAPs. The nucleotide binding metal (MeB) is coordinated at the active site prior to the catalytic metal (MeA). MeA coordination is only temporal, established just before and dissociated immediately after phosphodiester bond formation. We captured these elusive intermediates exploiting the slow enzymatic reaction in crystallo. These results demonstrate that the simple time-dependent soak-trigger-freeze X-ray crystallography offers a direct means for monitoring enzymatic reactions. Watching the bacteriophage N4 RNA polymerase transcription by time-dependent soak-trigger-freeze X-ray crystallography.,Basu RS, Murakami KS J Biol Chem. 2012 Dec 12. PMID:23235152 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also RNA polymerase 3D structures

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