Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation

Abasic sites are among the most abundant DNA lesions and interfere with DNA replication and transcription, but the mechanism of their action on transcription remains unknown. Here we applied a combined structural and biochemical approach for a comprehensive investigation of how RNA polymerase II (Pol II) processes an abasic site, leading to slow bypass of lesion. Encounter of Pol II with an abasic site involves two consecutive slow steps: insertion of adenine opposite a noninstructive abasic site (the A-rule), followed by extension of the 3′-rAMP with the next cognate nucleotide. Further studies provided structural insights into the A-rule: ATP is slowly incorporated into RNA in the absence of template guidance. Our structure revealed that ATP is bound to the Pol II active site, whereas the abasic site is located at an intermediate state above the Bridge Helix, a conserved structural motif that is cirtical for Pol II activity. The next extension step occurs in a template-dependent manner where a cognate substrate is incorporated, despite at a much slower rate compared with nondamaged template. During the extension step, neither the cognate substrate nor the template base is located at the canonical position, providing a structural explanation as to why this step is as slow as the insertion step. Taken together, our studies provide a comprehensive understanding of Pol II stalling and bypass of the abasic site in the DNA template.

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Structural basis of RNA polymerase II substrate specificity and catalysis

The FASEB Journal ◽

10.1096/fasebj.21.5.a656-c ◽

2007 ◽

Vol 21 (5) ◽

Author(s):

Dong Wang ◽

David A. Bushnell ◽

Kenneth D. Westover ◽

Craig D. Kaplan ◽

Roger D. Kornberg

Keyword(s):

Substrate Specificity ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Structural Basis

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Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7

10.1101/822460 ◽

2019 ◽

Author(s):

Fuzhou Ye ◽

Ioly Kotta-Loizou ◽

Milija Jovanovic ◽

Xiaojiao Liu ◽

David T. F. Dryden ◽

...

Keyword(s):

Rna Polymerase ◽

Protein A ◽

Restriction Enzymes ◽

Sigma Factors ◽

Structural Basis ◽

Bacteriophage T7 ◽

Transcription Inhibition ◽

Host Restriction ◽

Dna Mimicry ◽

Host Defence System

AbstractBacteriophage T7 infects Escherichia coli and evades the host defence system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics.Impact statementDNA mimicry Ocr protein, a well-studied T7 phage protein that inhibits host restriction enzymes, can also inhibit host transcription through competing with sigma factors in binding to RNA polymerase.

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Faculty Opinions recommendation of Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1000591.20907 ◽

2001 ◽

Author(s):

Michael Meisterernst

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Structural Basis

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