Indirect Effects of DNA Sequence on 434 Repressor-DNA Interactions

Abstract The CCR4-NOT transcriptional regulatory complex affects expression of a number of genes both positively and negatively. We report here that components of the CCR4-NOT complex functionally and physically interact with TBP and TBP-associated factors. First, mutations in CCR4-NOT components suppressed the his4-912δ insertion in a manner similar to that observed for the defective TBP allele spt15-122. Second, using modified HIS3 promoter derivatives containing specific mutations within the TATA sequence, we found that the NOT proteins were general repressors that disrupt TBP function irrespective of the DNA sequence. Third, increasing the dosage of NOT1 specifically inhibited the ability of spt15-122 to suppress the his4-912δ insertion but did not affect the Spt− phenotype of spt3 or spt10 at this locus. Fourth, spt3, spt8, and spt15-21 alleles (all involved in affecting interaction of SPT3 with TBP) suppressed ccr4 and caf1 defects. Finally, we show that NOT2 and NOT5 can be immunoprecipitated by TBP. NOT5 was subsequently shown to associate with TBP and TAFs and this association was dependent on the integrity of TFIID. These genetic and physical interactions indicate that one role of the CCR4-NOT proteins is to inhibit functional TBP-DNA interactions, perhaps by interacting with and modulating the function of TFIID.

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Encoding hierarchical assembly pathways of proteins with DNA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2106808118 ◽

2021 ◽

Vol 118 (40) ◽

pp. e2106808118

Author(s):

Oliver G. Hayes ◽

Benjamin E. Partridge ◽

Chad A. Mirkin

Keyword(s):

Dna Sequence ◽

Building Blocks ◽

Chemical Information ◽

Dna Interactions ◽

Sequence Design ◽

Hierarchical Assembly ◽

Direct Assembly ◽

Dna Sequence Design ◽

Dna Materials ◽

Assembly Pathways

The structural and functional diversity of materials in nature depends on the controlled assembly of discrete building blocks into complex architectures via specific, multistep, hierarchical assembly pathways. Achieving similar complexity in synthetic materials through hierarchical assembly is challenging due to difficulties with defining multiple recognition areas on synthetic building blocks and controlling the sequence through which those recognition sites direct assembly. Here, we show that we can exploit the chemical anisotropy of proteins and the programmability of DNA ligands to deliberately control the hierarchical assembly of protein–DNA materials. Through DNA sequence design, we introduce orthogonal DNA interactions with disparate interaction strengths (“strong” and “weak”) onto specific geometric regions of a model protein, stable protein 1 (Sp1). We show that the spatial encoding of DNA ligands leads to highly directional assembly via strong interactions and that, by design, the first stage of assembly increases the multivalency of weak DNA–DNA interactions that give rise to an emergent second stage of assembly. Furthermore, we demonstrate that judicious DNA design not only directs assembly along a given pathway but can also direct distinct structural outcomes from a single pathway. This combination of protein surface and DNA sequence design allows us to encode the structural and chemical information necessary into building blocks to program their multistep hierarchical assembly. Our findings represent a strategy for controlling the hierarchical assembly of proteins to realize a diverse set of protein–DNA materials by design.

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DNA Sequence Requirements for the Activation of 434 PRMTranscription by 434 Repressor

DNA and Cell Biology ◽

10.1089/104454900750019380 ◽

2000 ◽

Vol 19 (10) ◽

pp. 621-630 ◽

Cited By ~ 5

Author(s):

Jian Xu ◽

Gerald B. Koudelka

Keyword(s):

Dna Sequence ◽

Sequence Requirements ◽

434 Repressor

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The Role of the Minor Groove Substituents in Indirect Readout of DNA Sequence by 434 Repressor

Journal of Biological Chemistry ◽

10.1074/jbc.m212667200 ◽

2003 ◽

Vol 278 (15) ◽

pp. 12955-12960 ◽

Cited By ~ 24

Author(s):

Steven A. Mauro ◽

David Pawlowski ◽

Gerald B. Koudelka

Keyword(s):

Dna Sequence ◽

Minor Groove ◽

Indirect Readout ◽

434 Repressor

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The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation

Science Advances ◽

10.1126/sciadv.aaw9807 ◽

2019 ◽

Vol 5 (11) ◽

pp. eaaw9807 ◽

Cited By ~ 6

Author(s):

Qian Zhang ◽

Fengcai Wen ◽

Siqi Zhang ◽

Jiachuan Jin ◽

Lulu Bi ◽

...

Keyword(s):

Dna Sequence ◽

Single Molecule ◽

Dna Interactions ◽

Base Pairs ◽

Complementary Dna ◽

Interaction Site ◽

Protospacer Adjacent Motif ◽

Dna Loss ◽

Target Binding

Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a single-molecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.

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An improved ChEC-seq method accurately maps the genome-wide binding of transcription coactivators and sequence-specific transcription factors

10.1101/2021.02.12.430999 ◽

2021 ◽

Author(s):

Rafal Donczew ◽

Amélia Lalou ◽

Didier Devys ◽

Laszlo Tora ◽

Steven Hahn

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Dna Sequence ◽

Dna Interactions ◽

Experimental Conditions ◽

Genome Wide ◽

Protein Dna Interactions ◽

Cleavage Step ◽

Computational Analyses ◽

Active Genes

AbstractMittal and colleagues have raised questions about mapping transcription factor locations on DNA using the MNase-based ChEC-seq method (Mittal et al., 2021). Partly due to this concern, we modified the experimental conditions of the MNase cleavage step and subsequent computational analyses, resulting in more stringent conditions for mapping protein-DNA interactions (Donczew et al., 2020). The revised method (dx.doi.org/10.17504/protocols.io.bizgkf3w) answers questions raised by Mittal et al. and, without changing earlier conclusions, identified widespread promoter binding of the transcription coactivators TFIID and SAGA at active genes. The revised method is also suitable for accurately mapping the genome-wide locations of DNA sequence-specific transcription factors.

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