CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics

/pdf/crispr-cas12a-target-binding-unleashes-indiscriminate-single-3wl0bk4y7k.pdf

CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

Transcription initiation is the crucial focal point of gene expression in prokaryotes. The key players in this process, sigma factors (σs), associate with the catalytic core RNA polymerase to guide it through the essential steps of initiation: promoter recognition and opening, and synthesis of the first few nucleotides of the transcript. Here we recount the key advances in σ biology, from their discovery 45 years ago to the most recent progress in understanding their structure and function at the atomic level. Recent data provide important structural insights into the mechanisms whereby σs initiate promoter opening. We discuss both the housekeeping σs, which govern transcription of the majority of cellular genes, and the alternative σs, which direct RNA polymerase to specialized operons in response to environmental and physiological cues. The review concludes with a genome-scale view of the extracytoplasmic function σs, the most abundant group of alternative σs.

Bacterial Sigma Factors: A Historical, Structural, and Genomic Perspective

The regulated transcription of genes determines cell identity and function. Recent structural studies have elucidated mechanisms that govern the regulation of transcription by RNA polymerases during the initiation and elongation phases. Microscopy studies have revealed that transcription involves the condensation of factors in the cell nucleus. A model is emerging for the transcription of protein-coding genes in which distinct transient condensates form at gene promoters and in gene bodies to concentrate the factors required for transcription initiation and elongation, respectively. The transcribing enzyme RNA polymerase II may shuttle between these condensates in a phosphorylation-dependent manner. Molecular principles are being defined that rationalize transcriptional organization and regulation, and that will guide future investigations. Structural and microscopy studies of gene transcription underpin a model in which phosphorylation controls the shuttling of RNA polymerase II between promoter and gene-body condensates to regulate transcription initiation and elongation.

Organization and regulation of gene transcription.

In this Review, Browning and Busby describe the advances that have been made in recent years in understanding the molecular details of how transcription initiation is regulated to fine tune gene expression, highlighting factors that relate both to the RNA polymerase and to the promoter.

/pdf/local-and-global-regulation-of-transcription-initiation-in-4gsa0o2hgb.pdf

Local and global regulation of transcription initiation in bacteria

There is a continuous need for iterative cycles of antibiotic discovery and development to deal with the selection of resistant pathogens that emerge as therapeutic application of an antibiotic becomes widespread. A short golden age of antibiotic discovery from nature followed by a subsequent golden half century of medicinal chemistry optimization of existing molecular scaffolds emphasizes the need for new antibiotic molecular frameworks. We bring a molecule-centered perspective to the questions of where will new scaffolds come from, when will chemogenetic approaches yield useful new antibiotics and what existing bacterial targets merit contemporary re-examination.

Prospects for new antibiotics: a molecule-centered perspective

Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)–promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ A holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme.

/pdf/structural-basis-of-transcription-initiation-3v6nn0sloy.pdf

Mary X. Ho

Papers

Structural basis of transcription initiation.

Structures of RNA polymerase-antibiotic complexes