The interaction of RNA polymerase II with non-promoter DNA sites

Chandler, Donald Walt; Gralla, Jay D.

doi:10.1093/nar/9.22.6031

Cited by 9 publications

(6 citation statements)

References 23 publications

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“…DNA torsional strain was created which led to conformational transitions in certain polynucleotide sequences (Luchnik, 1980). Eukaryotic RNA polymerases prefer to transcribe TS DNA (Mandel and Chambon, 1974;Hossenlopp et al, 1974;Lescure et al, 1978;Lilley and Houghton, 1979;Chandler and Gralla, 1981). Thus, the detection of torsionally strained DNA molecules in a transcriptionally active fraction of SV40 minichromosomes is consistent with the above hypothesis.…”

Section: Biological Implicationsmentioning

confidence: 60%

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

Luchnik¹,

Bakayev²,

Zbarsky³

et al. 1982

The EMBO Journal

135

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After treatment of SV40 minichromosomes with DNA topoisomerase I, the superhelicity in the bulk of the DNA extracted from minichromosomes is known to remain unchanged. However, we found that the DNA extracted from a small fraction of SV40 minichromosomes (2‐5%), was almost completely relaxed, and covalently closed as shown by agarose gel electrophoresis. Thus, the DNA in these 2‐5% of SV40 minichromosomes was probably torsionally strained (TS). The proportion of such TS minichromosomes is close to the estimated proportion of transcriptionally active minichromosomes. The distribution of the TS minichromosomes in sucrose gradient coincided with the distribution of transcriptionally active complexes. Both sedimented faster than the majority of minichromosomes. Furthermore, after treatment with topoisomerase I the relaxed minichromosomes could be quantitatively separated from the bulk of material by recentrifugation in a sucrose gradient. A major part of the endogenous RNA polymerase activity was recovered in the relaxed fraction. These data suggest that TS‐minichromosomes correspond to transcriptionally active chromatin. After relaxation with topoisomerase I the TS minichromosomes lacked histones.

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Section: Biological Implicationsmentioning

confidence: 60%

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

Luchnik¹,

Bakayev²,

Zbarsky³

et al. 1982

The EMBO Journal

135

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“…RNA polymerase II and other RNA polymerases have a strong propensity to bind DNA nonspecifically (41), and RNA polymerase II has been observed within inactive genes (42). If such interactions lack TFIIB, as might be expected, then the inappropriately bound RNA polymerases would have difficulty producing RNA.…”

Section: Discussionmentioning

confidence: 97%

Control of the Timing of Promoter Escape and RNA Catalysis by the Transcription Factor IIB Fingertip

Tran

Gralla

2008

Journal of Biological Chemistry

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Transcription factor IIB (TFIIB) recruits RNA polymerase II to promoters and inserts a finger domain into its active site, with unknown consequences. Here we show that that the tip of this finger is important for two transcription initiation functions. First, TFIIB acts as a catalytic cofactor for initial RNA bond formation. It does so via a pair of fingertip aspartates that can bind magnesium, placing TFIIB within a family of proteins that insert finger domains to alter the catalytic functions of RNA polymerase. Second, the TFIIB fingertip mediates the timing of the release of TFIIB that is associated with appropriate promoter escape. These initiation requirements may assist in RNA quality control by minimizing functional synthesis when RNA polymerase becomes inappropriately associated with the genome without having been recruited there by TFIIB. TFIIB2 is a single-subunit protein that plays a central role in transcription by RNA polymerase II. It consists of three motifs with distinct structures and functions. The C terminus is composed of two cyclin repeats and is responsible for bringing TFIIB to promoters. It does so primarily by recognizing promoter-bound TATA-binding protein but also interacts with DNA sequences that flank the TATA box (1). A second motif is folded by association with a zinc atom (2). This binds a docking domain on RNA polymerase II (3) and recruits the enzyme to promoters to catalyze transcription initiation (4). A third motif is unstructured in solution but folds into a finger-like structure when bound to RNA polymerase (3). A number of functions have been suggested for this finger, mostly involving the escape by the recruited RNA polymerase (discussed below).TFIIB is unique among the general transcription factors in that it recycles during continuous transcription, i.e. TFIIF travels with the elongating RNA polymerase, and TATA-binding protein, TFIIH, and TFIIE largely remain bound to the promoter (5). Thus there can be a pioneer round of transcription that forms a scaffold upon which reinitiation can occur without requiring the reassembly of all factors from free solution. This allows transcription to occur at a facilitated rate (6). For each round of initiation, a new RNA polymerase must wait for the prior RNA polymerase to escape. This requires the recycling of TFIIB (7) by binding to the scaffold to recruit the next RNA polymerase. These initial steps of transcription initiation, involving initial bond formation, production of small RNAs, and disruption of contacts to the promoter, are collectively termed "escape" (8 -10).The finger domain of TFIIB has been proposed to play multiple roles in promoter escape. Both chemical probing (3) and structural studies (11) have shown that the finger penetrates the main channel of RNA polymerase II to approach the active site of the enzyme. From this location it could influence these early steps by interacting with the template DNA, the newly synthesized RNA, the catalytic center of the RNA polymerase, and perhaps TFIIF or other general transcr...

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“…Our RNAP sensitivity assay was inspired by the motivation to employ DNA origami structures as platforms for single-molecule analyses of transcription regulation at single-gene resolution. We anticipated that uncross-linked origami would be highly vulnerable to RNAPs, since origami, by definition, have numerous nicks and often have ssDNA modules, whereas RNAPs are known to nonspecifically initiate from nicks − and ssDNA . Sensitivity of nanostructures to viral RNAPs has been recently documented .…”

Section: Discussionmentioning

confidence: 99%

Functionalizing DNA Origami by Triplex-Directed Site-Specific Photo-Cross-Linking

Kalra,

Donnelly,

Singh

et al. 2024

J. Am. Chem. Soc.

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Here, we present a cross-linking approach to covalently functionalize and stabilize DNA origami structures in a one-pot reaction. Our strategy involves adding nucleotide sequences to adjacent staple strands, so that, upon assembly of the origami structure, the extensions form short hairpin duplexes targetable by psoralen-labeled triplex-forming oligonucleotides bearing other functional groups (pso-TFOs). Subsequent irradiation with UVA light generates psoralen adducts with one or both hairpin staples leading to site-specific attachment of the pso-TFO (and attached group) to the origami with ca. 80% efficiency. Bis-adduct formation between strands in proximal hairpins further tethers the TFO to the structure and generates "superstaples" that improve the structural integrity of the functionalized complex. We show that directing cross-linking to regions outside of the origami core dramatically reduces sensitivity of the structures to thermal denaturation and disassembly by T7 RNA polymerase. We also show that the underlying duplex regions of the origami core are digested by DNase I and thus remain accessible to read-out by DNA-binding proteins. Our strategy is scalable and cost-effective, as it works with existing DNA origami structures, does not require scaffold redesign, and can be achieved with just one psoralen-modified oligonucleotide.

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The interaction of RNA polymerase II with non-promoter DNA sites

Cited by 9 publications

References 23 publications

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

Control of the Timing of Promoter Escape and RNA Catalysis by the Transcription Factor IIB Fingertip

Functionalizing DNA Origami by Triplex-Directed Site-Specific Photo-Cross-Linking

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