Watching the Bacteriophage N4 RNA Polymerase Transcription by Time-dependent Soak-trigger-freeze X-ray Crystallography

Basu, Ritwika; Murakami, Katsuhiko

doi:10.1074/jbc.m112.387712

Cited by 35 publications

(40 citation statements)

References 38 publications

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“…However, the stopped-flow data with non-extendable DNA and Ca 2ϩ resulted in a poor fit to the same three-step model indicating that the non-covalent step is abolished under these conditions. This observation of the non-covalent step (step 3.1), taken together with the non-hydrolyzable analog data and the observation that step 3 (fingers closing) was similar with Ca 2ϩ and Mg 2ϩ , may be a result of a rearrangement of the active site (Asp-256, Asp-190, and Asp-192) during the binding of metal A, as recently suggested (23,34,39,42,55). Other possibilities include local rearrangement of active site side chains as recently suggested (44).…”

Section: Scheme 2 Biochemical Pathway Model For Kintek Explorer Simumentioning

confidence: 78%

Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β

Towle-Weicksel¹,

Dalal²,

Sohl

et al. 2014

Journal of Biological Chemistry

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Background: DNA Pol ␤ participates in base excision repair by choosing correct dNTP to fill single-nucleotide gaps in DNA. Results: Pol ␤ experiences a non-covalent step with correct dNTP selection. Conclusion: Correct and incorrect dNTP incorporation by Pol ␤ are different. Significance: FRET-based system of Pol ␤ elucidates a mechanism of substrate choice necessary for understanding the molecular basis of human disease.

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Section: Scheme 2 Biochemical Pathway Model For Kintek Explorer Simumentioning

confidence: 78%

Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β

Towle-Weicksel¹,

Dalal²,

Sohl

et al. 2014

Journal of Biological Chemistry

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“…Triphosphate intensity reduction showed that catalysis was complete shortly after 10 minutes. Basu and Murakami 40 used the Raman kinetic data to flash freeze and solve the structures of the N4 crystals at time points between the 0 to 10 minutes of soak in. The predictions from Raman crystallography were borne out, they were able to obtain high quality X-ray structures for intermediates during the formation of the covalent nucleic acid backbone bond.…”

Section: Discussionmentioning

confidence: 99%

Following DNA Chain Extension and Protein Conformational Changes in Crystals of a Y-Family DNA Polymerase via Raman Crystallography

et al. 2013

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Y-family DNA polymerases are known to bypass DNA lesions in vitro and in vivo. Sulfolobus solfataricus DNA polymerase (Dpo4) was chosen as a model Y-family enzyme for investigating the mechanism of DNA synthesis in single crystals. Crystals of Dpo4 in complexes with DNA (the binary complex) in the presence or absence of an incoming nucleotide were analyzed by Raman microscopy. 13C, 15N labeled d*CTP, or unlabeled dCTP, were soaked into the binary crystals with G as the templating base. In the presence of the catalytic metal ions, Mg2+ or Mn2+, nucleotide incorporation was detected by the disappearance of the triphosphate band of dCTP and the retention of C* modes in the crystal following soaking out of noncovalently bound C(or *C)TP. The addition of the second coded base, thymine, was observed by adding cognate dTTP to the crystal following single d*CTP addition. Adding these two bases caused visible damage to the crystal possibly caused by protein and/or DNA conformational change within the crystal. When d*CTP is soaked into the Dpo4 crystal in the absence of Mn2+ or Mg2+, the primer extension reaction did not occur; instead a ternary protein/template/d*CTP complex was formed. In the Raman difference spectra of both binary and ternary complexes, in addition to the modes of d(*C)CTP, features appear due to ring modes from the template/primer bases being perturbed and from the DNA backbone, as well as from perturbed peptide and amino acid side chain modes. These effects are more pronounced in the ternary than in the binary complex. Using standardized Raman intensities followed as a function of time C(*C)TP population in the crystal maximized at about 20 min. These remained unchanged in the ternary complex but declined in the binary complexes as chain incorporation occurred.

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“…The in crystallo transcription system can be used to study RNA transcription by Raman crystallography and time-resolved trigger-freeze crystallography. Analogous to the studies performed on single-subunit RNAPs (41,42), these new experimental approaches may allow to trace events in not only initial transcription but also during the NTP addition cycle in cellular RNAPs.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme

Basu

Warner

Molodtsov

et al. 2014

Journal of Biological Chemistry

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163

212

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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.

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Watching the Bacteriophage N4 RNA Polymerase Transcription by Time-dependent Soak-trigger-freeze X-ray Crystallography

Cited by 35 publications

References 38 publications

Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β

Fluorescence Resonance Energy Transfer Studies of DNA Polymerase β

Following DNA Chain Extension and Protein Conformational Changes in Crystals of a Y-Family DNA Polymerase via Raman Crystallography

Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme

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