Author response: CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im–induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im–induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides–dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im–induced transcriptional arrest.

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CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex

eLife ◽

10.7554/elife.08505 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 38

Author(s):

Brian Bae ◽

James Chen ◽

Elizabeth Davis ◽

Katherine Leon ◽

Seth A Darst ◽

...

Keyword(s):

Transcription Initiation ◽

Minor Groove ◽

Initiation Complex ◽

Regulate Gene Expression ◽

E Coli ◽

Transcription Bubble ◽

Promoter Complex ◽

Transcription Initiation Complex ◽

A Minor

A key point to regulate gene expression is at transcription initiation, and activators play a major role. CarD, an essential activator in Mycobacterium tuberculosis, is found in many bacteria, including Thermus species, but absent in Escherichia coli. To delineate the molecular mechanism of CarD, we determined crystal structures of Thermus transcription initiation complexes containing CarD. The structures show CarD interacts with the unique DNA topology presented by the upstream double-stranded/single-stranded DNA junction of the transcription bubble. We confirm that our structures correspond to functional activation complexes, and extend our understanding of the role of a conserved CarD Trp residue that serves as a minor groove wedge, preventing collapse of the transcription bubble to stabilize the transcription initiation complex. Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter complexes, explaining the need for CarD.

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Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder

Nucleic Acids Research ◽

10.1093/nar/25.13.2657 ◽

1997 ◽

Vol 25 (13) ◽

pp. 2657-2660 ◽

Cited By ~ 120

Author(s):

I Afonina

Keyword(s):

Minor Groove ◽

Minor Groove Binder ◽

A Minor

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Fine structure of E. coli RNA polymerase-promoter interactions: alpha subunit binding to the UP element minor groove

Genes & Development ◽

10.1101/gad.870001 ◽

2001 ◽

Vol 15 (5) ◽

pp. 491-506 ◽

Cited By ~ 68

Author(s):

W. Ross

Keyword(s):

Fine Structure ◽

Rna Polymerase ◽

Minor Groove ◽

Alpha Subunit ◽

E Coli ◽

Rna Polymerase Promoter

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A Minor Groove Binding Copper-Phenanthroline Conjugate Produces Direct Strand Breaks via β-Elimination of 2-Deoxyribonolactone

Journal of the American Chemical Society ◽

10.1021/ja026970z ◽

2002 ◽

Vol 124 (31) ◽

pp. 9062-9063 ◽

Cited By ~ 64

Author(s):

Brian C. Bales ◽

Marguerite Pitié ◽

Bernard Meunier ◽

Marc M. Greenberg

Keyword(s):

Minor Groove ◽

Minor Groove Binding ◽

Groove Binding ◽

Strand Breaks ◽

A Minor

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Mechanism of RNA polymerase II stalling by DNA alkylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1706592114 ◽

2017 ◽

Vol 114 (46) ◽

pp. 12172-12177 ◽

Cited By ~ 8

Author(s):

Stefano Malvezzi ◽

Lucas Farnung ◽

Claudia M. N. Aloisi ◽

Todor Angelov ◽

Patrick Cramer ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Anticancer Agents ◽

Excision Repair ◽

Minor Groove ◽

Dna Alkylation ◽

Drug Induced ◽

Rna Pol Ii ◽

Pol Ii

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

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