ABSTRACT
Transcription activation is a crucial step of regulation during transcription initiation and a classic check point in response to different stimuli and stress factors. The Escherichia coli NarL is a nitrate-responsive global transcription factor that controls the expression of nearly 100 genes. However, the molecular mechanism of NarL-mediated transcription activation is not well defined. Here we present a cryo-EM structure of NarL-dependent transcription activation complex (TAC) assembled on the yeaR promoter at 3.2 Å resolution. Our structure shows that the NarL dimer binds at the -43.5 site of the promoter DNA with its C-terminal domain (CTD) not only binding to the DNA but also making interactions with RNA polymerase subunit alpha CTD (αCTD). The key role of these NarL-mediated interactions in transcription activation was further confirmed by in vivo and in vitro transcription assays. Additionally, the NarL dimer binds DNA in a different plane from that observed in the structure of class II TACs. Unlike the canonical class II activation mechanism, NarL does not interact with σ4, while RNAP αCTD is bound to DNA on the opposite side of NarL. Our findings provide a structural basis for detailed mechanistic understanding of NarL-dependent transcription activation on yeaR promoter and reveal a potentially novel mechanism of transcription activation.
Subject(s)
Escherichia coli Proteins , Nitrates , Transcriptional Activation , Bacterial Proteins/metabolism , DNA/genetics , DNA/metabolism , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Nitrates/metabolismABSTRACT
Transcription activation is an important checkpoint of regulation of gene expression which occurs in response to different intracellular and extracellular signals. The key elements in this signal transduction process are transcription activators, which determine when and how gene expression is activated. Recent structural studies on a considerable number of new transcription activation complexes (TACs) revealed the remarkable mechanistic diversity of transcription activation mediated by different factors, necessitating a review and re-evaluation of the transcription activation mechanisms. In this review, we present a comprehensive summary of transcription activation mechanisms and propose a new, elaborate, and systematic classification of transcription activation mechanisms, primarily based on the structural features of diverse TAC components.
Subject(s)
Bacterial Proteins , DNA-Directed RNA Polymerases , Transcriptional Activation , Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Sigma Factor/metabolism , Promoter Regions, Genetic , Bacteria/genetics , Bacteria/metabolism , Gene Expression Regulation, Bacterial , Transcription, GeneticABSTRACT
CRISPR-Cas is a prokaryotic adaptive immune system, classified into six different types, each characterised by a signature protein. Type III systems, classified based on the presence of a Cas10 subunit, are rather diverse multi-subunit assemblies with a range of enzymatic activities and downstream ancillary effectors. The broad array of current biotechnological CRISPR applications is mainly based on proteins classified as Type II, however recent developments established the feasibility and efficacy of multi-protein Type III CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes. The crenarchaeon Saccharolobus solfataricus has two type III system subtypes (III-B and III-D). Here, we report the cryo-EM structure of the Csm Type III-D complex from S. solfataricus (SsoCsm), which uses CRISPR RNA to bind target RNA molecules, activating the Cas10 subunit for antiviral defence. The structure reveals the complex organisation, subunit/subunit connectivity and protein/guide RNA interactions of the SsoCsm complex, one of the largest CRISPR effectors known.
