4FF3

N4 mini-vRNAP transcription initiation complex, 3 min after soaking GTP, ATP and Mn

Summary for 4FF3

• Entry DOI: 10.2210/pdb4ff3/pdb
• Related: 4ff1 4ff2 4ff4
• Descriptor: Virion RNA polymerase, Bacteriophage N4 P2 promoter, ADENOSINE-5'-TRIPHOSPHATE, ... (7 entities in total)
• Functional Keywords: rna polymerase, transferase-dna complex, transferase/dna
• Biological source: Enterobacteria phage N4; More
• Total number of polymer chains: 4
• Total formula weight: 269601.42

Authors

Murakami, K.S.,Basu, R.S. (deposition date: 2012-05-30, release date: 2012-12-12, Last modification date: 2024-02-28)

Primary citation

Basu, R.S.,Murakami, K.S.
Watching the Bacteriophage N4 RNA Polymerase Transcription by Time-dependent Soak-trigger-freeze X-ray Crystallography.
J.Biol.Chem., 288:3305-3311, 2013

PubMed Abstract: The challenge for structural biology is to understand atomic-level macromolecular motions during enzymatic reaction. 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 which allow watching the nucleotide and metal bindings and the phosphodiester bond formation in a time perspective. Our study provides the temporal order of substrate assembly and metal co-factor binding at the active site of enzyme which completes our understanding of the two-metal-ion mechanism and fidelity mechanism in single-subunit RNAPs. The nucleotide-binding metal (Me(B)) is coordinated at the active site prior to the catalytic metal (Me(A)). Me(A) 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.

PubMed: 23235152
DOI: 10.1074/jbc.M112.387712

Experimental method

X-RAY DIFFRACTION (1.997 Å)