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Abstract: Tumor-suppressor let-7 pre-microRNAs (miRNAs) are regulated by terminal uridylyltransferases TUT7 and TUT4 that either promote let-7 maturation by adding a single uridine nucleotide to the pre-miRNA 3′ end or mark them for degradation by the addition of multiple uridines. Oligo-uridylation is increased in cells by enhanced TUT7/4 expression and especially by the RNA-binding pluripotency factor LIN28A. Using cryogenic electron microscopy, we captured high-resolution structures of active forms of TUT7 alone, of TUT7 plus pre-miRNA and of both TUT7 and TUT4 bound with pre-miRNA and LIN28A. Our structures reveal that pre-miRNAs engage the enzymes in fundamentally different ways depending on the presence of LIN28A, which clamps them onto the TUTs to enable processive 3′ oligo-uridylation. This study reveals the molecular basis for mono- versus oligo-uridylation by TUT7/4, as determined by the presence of LIN28A, and thus their mechanism of action in the regulation of cell fate and in cancer.

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Abstract: Most healthy people carry bacteria-infecting viruses called crassviruses in their gut, which may help to maintain the gut microbiome. However, scientists know little about this group of viruses, including their structure. This led researchers at the University of York to visit the electron Bio-Imaging Centre (eBIC) at Diamond Light Source where they captured images of these elusive viruses for the first time. Using high-voltage cryo-electron microscopes at eBIC, they overcame challenges posed by these large virus assemblies, resolving their structure to a near-atomic resolution. They found that the virus consists of a “head” region that stores viral DNA and proteins. The head is connected to a “tail” that injects these contents into bacteria, allowing virus replication to take place inside microbial cells. Additionally, they discovered a muzzle that prevents DNA and proteins from leaking out through the tail. A never-before-seen “crass” fold was spotted in the tail that the team hypothesised loosens and tightens the muzzle to control the flow of viral material into the bacterium. This study, published in Nature, offers a first glimpse at an underexplored family of widespread viruses and provides insight into their lifecycle.

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Abstract: Amongst the major types of archaeal filaments, several have been shown to closely resemble bacterial homologues of the Type IV pili (T4P). Within Sulfolobales, member species encode for three types of T4P, namely the archaellum, the UV-inducible pilus system (Ups) and the archaeal adhesive pilus (Aap). Whereas the archaellum functions primarily in swimming motility, and the Ups in UV-induced cell aggregation and DNA-exchange, the Aap plays an important role in adhesion and twitching motility. Here, we present a cryoEM structure of the Aap of the archaeal model organism Sulfolobus acidocaldarius. We identify the component subunit as AapB and find that while its structure follows the canonical T4P blueprint, it adopts three distinct conformations within the pilus. The tri-conformer Aap structure that we describe challenges our current understanding of pilus structure and sheds new light on the principles of twitching motility.

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Abstract: Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins1,2,3. Although some transposases act alone, many rely on dedicated AAA+ ATPase subunits that regulate site selectivity and catalytic function through poorly understood mechanisms. Using IS21 as a model transposase system, we show how an ATPase regulator uses nucleotide-controlled assembly and DNA deformation to enable structure-based site selectivity, transposase recruitment, and activation and integration. Solution and cryogenic electron microscopy studies show that the IstB ATPase self-assembles into an autoinhibited pentamer of dimers that tightly curves target DNA into a half-coil. Two of these decamers dimerize, which stabilizes the target nucleic acid into a kinked S-shaped configuration that engages the IstA transposase at the interface between the two IstB oligomers to form an approximately 1 MDa transpososome complex. Specific interactions stimulate regulator ATPase activity and trigger a large conformational change on the transposase that positions the catalytic site to perform DNA strand transfer. These studies help explain how AAA+ ATPase regulators—which are used by classical transposition systems such as Tn7, Mu and CRISPR-associated elements—can remodel their substrate DNA and cognate transposases to promote function.

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Abstract: Transposition encompasses a range of biological processes in which a DNA segment can move between different genomic locations using enzymes called transposases. This ubiquitous process can cause mutations in the host genome, drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins. Transposition must be carefully regulated to minimize harm to the host and prevent deleterious DNA breaks. Numerous families of transposases rely on dedicated AAA+ ATPases that regulate both target selectivity and catalytic function. Nevertheless, the molecular mechanisms that these proteins use are still poorly understood. In this work we use the streamlined IS21 transposon as a model system in an attempt to shed light on these questions. Besides being a particularly widespread transposable element, IS21 codifies only two proteins: the transposase (IstA) and an essential regulatory ATPase (IstB). Using cryo-EM we show that the IstB ATPase self-assembles into a pentamer-of-dimers in a nucleotide-dependent fashion. IstB employs dedicated N-terminal domains to adopt an autoinhibited configuration that binds target DNA in an atypical way, introducing an overall 1800 bend onto it. Subsequent cryo-EM study of the complexes formed by IstB and the IstA transposase show that these IstB decamers are able to further dimerize to enforce an S-shape on the target DNA and form a landing pad for IstA transposase bound to the IS21 transposon ends. Specific protein-protein interactions activate the ATPase activity of IstB, remodelling the IstA-proximal nucleotide binding pockets. Furthermore, interaction with IstB enforces a marked conformational change on IstA, from the previously described intertwined plectoneme, to an open scissor-shaped configuration. The remodelling of the transposase positions the catalytic sites with an adequate geometry to perform the strand- transfer reaction. Preliminary single-molecule experiments provided further information into the DNA deformation caused by IstB and the kinetics of the transposition reaction. These results reveal how both proteins mutually activate each other, allowing us to propose a model for IS21 transposition, and drawing similarities and differences with related transposable elements to show how an ATPase-regulator uses nucleotide-controlled assembly and DNA deformation to enable site selectivity, transposase recruitment/activation, and integration. These studies help explain how AAA+ ATPase regulators – which are used by classic transposition systems such as Tn7, Mu, and CRISPR-associated elements– can remodel their substrate DNA and cognate transposases to perform their function.