Aminoacyl chain translocation catalysed by a type II thioesterase domain in an unusual non-ribosomal peptide synthetase

Non-Ribosomal Peptide Synthetases (NRPSs) assemble a diverse range of natural products with important applications in both medicine and agriculture. They consist of several multienzyme subunits that must interact with each other in a highly controlled manner to facilitate efficient chain transfer, thus ensuring biosynthetic fidelity. Several mechanisms for chain transfer are known for NRPSs, promoting structural diversity. Herein, we report the first biochemically characterized example of a type II thioesterase (TEII) domain capable of catalysing aminoacyl chain transfer between thiolation (T) domains on two separate NRPS subunits responsible for installation of a dehydrobutyrine moiety. Biochemical dissection of this process reveals the central role of the TEII-catalysed chain translocation event and expands the enzymatic scope of TEII domains beyond canonical (amino)acyl chain hydrolysis. The apparent co-evolution of the TEII domain with the NRPS subunits highlights a unique feature of this enzymatic cassette, which will undoubtedly find utility in biosynthetic engineering efforts.

The Processivity of Telomerase: Insights from Kinetic Simulations and Analyses

Telomerases are moderately processive reverse transcriptases that use an integral RNA template to extend the 3′ end of linear chromosomes. Processivity values, defined as the probability of extension rather than dissociation, range from about 0.7 to 0.99 at each step. Consequently, an average of tens to hundreds of nucleotides are incorporated before the single-stranded sDNA product dissociates. The RNA template includes a six nucleotide repeat, which must be reset in the active site via a series of translocation steps. Nucleotide addition associated with a translocation event shows a lower processivity (repeat addition processivity, RAP) than that at other positions (nucleotide addition processivity, NAP), giving rise to a characteristic strong band every 6th position when the product DNA is analyzed by gel electrophoresis. Here, we simulate basic reaction mechanisms and analyze the product concentrations using several standard procedures to show how the latter can give rise to systematic errors in the processivity estimate. Complete kinetic analysis of the time course of DNA product concentrations following a chase with excess unlabeled DNA primer (i.e., a pulse-chase experiment) provides the most rigorous approach. This analysis reveals that the higher product concentrations associated with RAP arise from a stalling of nucleotide incorporation reaction during translocation rather than an increased rate constant for the dissociation of DNA from the telomerase.

Rapid diffusion of large TatA complexes detected using single particle tracking microscopy

The twin-arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane of most bacteria and archaea. TatA, which contains a single membrane-spanning helix, is believed to be responsible for the actual translocation. According to the prevalent model, multiple TatA subunits form a transient protein-conducting pore, which disassembles after each translocation event. An alternative model exists, in which TatA proteins locally weaken the lipid bilayer to translocate folded proteins. Here, we imaged eGFP-fused TatA expressed from the genome in live E. coli cells. Images showed TatA occuring both in highly mobile monomers or small oligomers and in large, stable complexes that do not dissociate. Single-particle tracking revealed that large TatA complexes switch between fast and slow diffusion. The fast diffusion is too fast for a transmembrane protein complex consisting of multiple TatA monomers. In line with recent data on rhomboid proteases, we propose that TatA complexes switch between a slowly diffusing transmembrane conformation and a rapidly diffusing membrane-disrupting state that enables folded proteins to cross the membrane, in accordance with the membrane-weakening model.

Insights into substrate-mediated assembly of the chloroplast TAT receptor complex

The Twin Arginine Transport (TAT) system translocates fully folded proteins across the thylakoid membrane in the chloroplast (cp) and the cytoplasmic membrane of bacteria. In chloroplasts, cpTAT transport is achieved by three components: Tha4, Hcf106, and cpTatC. Hcf106 and cpTatC function as the substrate recognition/binding complex while Tha4 is thought to play a significant role in forming the translocation pore. Recent studies challenged this idea by suggesting that cpTatC-Hcf106-Tha4 function together in the active translocase. Here, we have mapped the inter-subunit contacts of cpTatC-Hcf106 during the resting state and built a cpTatC-Hcf106 structural model based on our crosslinking data. In addition, we have identified a substrate-mediated reorganization of cpTatC-Hcf106 contact sites during active substrate translocation. The proximity of Tha4 to the cpTatC-Hcf106 complex was also identified. Our data suggest a model for cpTAT function in which the transmembrane helices of Hcf106 and Tha4 may each contact the fifth transmembrane helix of cpTatC while the insertion of the substrate signal peptide may rearrange the cpTatC-Hcf106-Tha4 complex and initiate the translocation event.One sentence summaryProtein subunits of the thylakoidal twin arginine transport complex function together during substrate recognition and translocase assembly.

A 6.5kb intergenic structural variation enhances P450-mediated resistance to pyrethroids in malaria vectors lowering bed net efficacy

Elucidating the complex evolutionary armory that mosquitoes deploy against insecticides is crucial to maintain the effectiveness of insecticide-based interventions. Here, we deciphered the role of a 6.5kb structural variation (SV) in driving cytochrome P450-mediated pyrethroid resistance in the malaria vector, Anopheles funestus. Whole genome pooled sequencing detected an intergenic 6.5kb SV between duplicated CYP6P9a/b P450s in pyrethroid resistant mosquitoes through a translocation event. Promoter analysis revealed a 17.5-fold higher activity (P<0.0001) for the SV-carrying fragment than the SV-free one. qRT-PCR expression profiling of CYP6P9a/b for each SV genotype supported its role as an enhancer since SV+/SV+ homozygote mosquitoes had significantly greater expression for both genes than heterozygotes SV+/SV- (1.7-2-fold) and homozygotes SV-/SV- (4-5-fold). Designing a PCR assay revealed a strong association between this SV and pyrethroid resistance (SV+/SV+ vs SV-/SV-; OR=2079.4, P=<0.001). The 6.5kb SV is present at high frequency in southern Africa (80-100%) but absent in East/Central/West Africa. Experimental hut trials revealed that homozygote SV mosquitoes had significantly greater chance to survive exposure to pyrethroid-treated Nets (OR 27.7; P < 0.0001) and to blood feed than susceptible. Furthermore, triple homozygote resistant (SV+/CYP6P9a_R/CYP6P9b_R) exhibit a higher resistance level leading to a far superior ability to survive exposure to nets than triple susceptible mosquitoes, revealing a strong additive effect. This study highlights the important role of structural variations in the development of insecticide resistance in malaria vectors and their detrimental impact on the effectiveness of pyrethroid-based nets.

Reconstruction of Ewing Sarcoma Developmental Context from Mass-Scale Transcriptomics Reveals Characteristics of EWSR1-FLI1 Permissibility

Ewing sarcoma is an aggressive pediatric cancer of enigmatic cellular origins typically resulting from a single translocation event t (11; 22) (q24; q12). The resulting fusion gene, EWSR1-FLI1, is toxic or unstable in most primary tissues. Consequently, attempts to model Ewing sarcomagenesis have proven unsuccessful thus far, highlighting the need to identify the cellular features which permit stable EWSR1-FLI1 expression. By re-analyzing publicly available RNA-Sequencing data with manifold learning techniques, we uncovered a group of Ewing-like tissues belonging to a developmental trajectory between pluripotent, neuroectodermal, and mesodermal cell states. Furthermore, we demonstrated that EWSR1-FLI1 expression levels control the activation of these developmental trajectories within Ewing sarcoma cells. Subsequent analysis and experimental validation demonstrated that the capability to resolve R-loops and mitigate replication stress are probable prerequisites for stable EWSR1-FLI1 expression in primary tissues. Taken together, our results demonstrate how EWSR1-FLI1 hijacks developmental gene programs and advances our understanding of Ewing sarcomagenesis.

Microfluidic device for coupling isotachophoretic sample focusing with nanopore single-molecule sensing

Electrokinetic focusing of analytes to a solid-state nanopore enhances the translocation event rate, enabling rapid nanopore sensing at low analyte concentrations.

Molecular mechanism and history of non-sense to sense evolution of antifreeze glycoprotein gene in northern gadids

A fundamental question in evolutionary biology is how genetic novelty arises. De novo gene birth is a recently recognized mechanism, but the evolutionary process and function of putative de novo genes remain largely obscure. With a clear life-saving function, the diverse antifreeze proteins of polar fishes are exemplary adaptive innovations and models for investigating new gene evolution. Here, we report clear evidence and a detailed molecular mechanism for the de novo formation of the northern gadid (codfish) antifreeze glycoprotein (AFGP) gene from a minimal noncoding sequence. We constructed genomic DNA libraries for AFGP-bearing and AFGP-lacking species across the gadid phylogeny and performed fine-scale comparative analyses of theAFGPgenomic loci and homologs. We identified the noncoding founder region and a nine-nucleotide (9-nt) element therein that supplied the codons for one Thr-Ala-Ala unit from which the extant repetitive AFGP-coding sequence (cds) arose through tandem duplications. The latent signal peptide (SP)-coding exons were fortuitous noncoding DNA sequence immediately upstream of the 9-nt element, which, when spliced, supplied a typical secretory signal. Through a 1-nt frameshift mutation, these two parts formed a single read-through open reading frame (ORF). It became functionalized when a putative translocation event conferred the essentialcispromoter for transcriptional initiation. We experimentally proved that all genic components of the extant gadidAFGPoriginated from entirely nongenic DNA. The gadidAFGPevolutionary process also represents a rare example of the proto-ORF model of de novo gene birth where a fully formed ORF existed before the regulatory element to activate transcription was acquired.

They don’t live forever: How life history data and encounter probability help to assess success of Muscardinus avellanarius translocations (Rodentia: Gliridae)

The hazel dormouse (Muscardinus avellanarius) is a European Protected Species and for this reason, hazel dormice are protected from deliberate killing, injury or disturbance and its sites and resting places are also protected. During development projects impacts on hazel dormouse individuals and populations should be avoided. If avoidance is not possible measures of mitigation and compensation have to be implemented. In many cases the only suitable measure to prevent disturbance, killing or injury of individuals is the translocation of hazel dormice to another suitable habitat. The success of translocations has so far been rarely documented. To assess the success of translocations, the natural mortality of hazel dormice has to be considered as well as the likelihood of finding specific individuals during the proposed action. How these data affect the assessment of translocation success is calculated based on published data on seasonal survival rates of different cohorts and of unpublished monthly encounter probabilities of a population of marked animals. Depending on the time between the translocation event and the subsequent monitoring controls the number of hazel dormice likely to be alive can be low. For this reason, success cannot be evaluated with our method if the sample size is too small.