Septins constitute a family of guanine nucleotidebinding proteins that were first discovered in the yeast Saccharomyces cerevisiae but are also present in many other eukaryotes. In yeast they congregate at the bud neck and are required for cell division. Their function in metazoan cells is uncertain, but they have been implicated in exocytosis and cytokinesis. Septins have been purified from cells as hetero-oligomeric filaments, but their mechanism of assembly is unknown. Further studies have been limited by the difficulty in expressing functional septin proteins in bacteria. We now show that stable, soluble septin heterodimers can be produced by co-expression from bicistronic vectors in bacteria and that the co-expression of three septins results in their assembly into filaments. Pre-assembled dimers and trimers bind guanine nucleotide and show a slow GTPase activity. The assembly of a heterodimer from monomers in vitro is accompanied by GTP hydrolysis. Borg3, a downstream effector of the Cdc42 GTPase, binds specifically to a septin heterodimer composed of Sept6 and Sept7 and to the Sept2/6/7 trimer, but not to septin monomers or to other heterodimers. Septins associate through their C-terminal coiled-coil domains, and Borg3 appears to recognize the interface between these domains in Sept6 and Sept7.

We analyzed mutations and gene copy number changes in nontumor, IEN, and ESCC samples, collected from 70 patients. IEN and ESCCs each had similar mutations and markers of genomic instability, including apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like. Genomic changes observed in precancerous lesions might be used to identify patients at risk for ESCC.

Six RNA (pRNA) molecules form a hexamer, via handin-hand interaction, to gear bacterial virus phi29 DNA translocation machinery. Here we report the pathway and the conditions for the hexamer formation. Stable pRNA dimers and trimers were assembled in solution, isolated from native gels, and separated by sedimentation, providing a model system for the study of RNA dimers and trimers in a protein-free environment. Cryoatomic force microscopy revealed that monomers displayed a ߜ outline, dimers exhibited an elongated shape, and trimers formed a triangle. Dimerization of pRNA was promoted by a variety of cations including spermidine, whereas procapsid binding and DNA packaging required specific divalent cations, including Mg 2؉ , Ca 2؉ , and Mn 2؉ . Both the tandem and fused pRNA dimers with complementary loops designed to form even-numbered rings were active in DNA packaging, whereas those without complementary loops were inactive. We conclude that dimers are the building blocks of the hexamer, and the pathway of building a hexamer is: dimer 3 tetramer 3 hexamer. The Hill coefficient of 2.5 suggests that there are three binding sites with cooperative binding on the surface of the procapsid. The two interacting loops played a key role in recruiting the incoming dimer, whereas the procapsid served as the foundation for hexamer assembly.Multimerization of RNA molecules plays diverse roles in various biological systems. Dimerization of retrovirus RNA is believed to govern the essential steps of the retroviral life cycle, including translation, reverse transcription, RNA encapsidation, and virion assembly (1-5). During the early events of pre-mRNA splicing, there are several types of interactions through a network of RNA-RNA, RNA-protein, and proteinprotein contacts (6 -10). In addition, RNA-RNA interactions are involved in the cleavage of pre-tRNA by RNase P (11-13) and genetic regulation in bacteria (14, 15), eukaryotes (16), plants (17), mammals (18), and plasmids (19).Recently, we reported that phi29 RNA interacts hand-inhand to form hexamers to gear a DNA translocation machinery (20 -27) that uses ATP as the energy source (28). Such loop-loop interaction of phi29 pRNA is different from pseudoknots (29, 30) and kissing loops (1, 3-5, 31-34) that have been characterized previously. Pseudoknots involve the intramolecular interactions within one single molecule, and kissing loops involve the interaction of two self-complementary loops to form a dimer (1-5, 36). Since phi29 pRNAs form closed rings, the intermolecular interaction of pRNAs must require that each RNA molecule contribute one loop to pair with an alternate loop of the next pRNA. The key feature of the "hand-in-hand model" is that multiple RNAs interact via alternating interlocking loops to form a closed ring. Such hand-in-hand loop-loop interactions may also play an important role in other systems as well. For example, RNA-RNA interaction via alternating loops has also been reported for bicoid mRNA in Drosophila embryos (37). We speculated that the mechanism...

Using atomic force microscopy (AFM) in aqueous solution, we show that the surface structure of the oligomeric GroES can be obtained up to 10 A resolution. The seven subunits of the heptamer were well resolved without image averaging. The overall dimension of the GroES heptamer was 8.4+0.4 nm in diameter and 3.0+0.3 nm high. However, the AFM images further suggest that there is a central protrusion of 0.8+0.2 nm high and 4.5+0.4 nm in diameter on one side of GroES which displays a profound seven-fold symmetry. It was found that GroEL could not bind to the adsorbed GroES in the presence of AMP-PNP and Mg 2+, suggesting that the side of GroES with the central protrusion faces away from the GroEL lumen, because only one side of GroES was observed under these conditions. Based on the results from both electron and atomic force microscopy, a surface model for the GroES is proposed.Key words: Atomic force microscopy; GroES; GroEL; Resolution subunits were not discernible [18]. Because of the variable conformations observed by EM, it was even suggested that GroES could have a flexible structure or a symmetry other than the seven-fold [18]. Since the atomic force microscopy (AFM) has been shown to be capable of obtaining high resolution surface structures of several oligomeric bacterial proteins (for recent reviews, see [24][25][26]), we have applied this method to determine the surface structure of GroES under aqueous solutions. We show that the subunit structure can be clearly resolved in the AFM images without image processing/averaging. Surface structures beyond the subunits were also resolved, demonstrating a surface resolution of 10 A or so, which is much higher than that from EM images. In combination with the results from EM, a model for the GroES is also proposed. Materials and methods

The outbreak of COVID-19 caused by a novel Coronavirus (termed SARS-CoV-2) has spread to over 210 countries around the world. Currently, reverse transcription quantitative qPCR (RT-qPCR) is used as the gold standard for diagnosis of SARS-CoV-2. However, the sensitivity of RT-qPCR assays of pharyngeal swab samples are reported to vary from 30% to 60%. More accurate and sensitive methods are urgently needed to support the quality assurance of the RT-qPCR or as an alternative diagnostic approach. A reverse transcription digital PCR (RT-dPCR) method was established and evaluated. To explore the feasibility of RT-dPCR in diagnostic of SARS-CoV-2, a total of 196 clinical pharyngeal swab samples from 103 suspected patients, 77 close contacts and 16 supposed convalescents were analyzed by RT-qPCR and then measured by the proposed RT-dPCR. For the 103 fever suspected patients, 19 (19/25) negative and 42 (42/49) equivocal tested by RT-qPCR were positive according to RT-dPCR. The sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR. For 29 close contacts (confirmed by additional sample and clinical follow up), 16 (16/17) equivocal and 1 negative tested by RT-qPCR were positive according to RT-dPCR, which is implying that the RT-qPCR is missing a lot of asymptomatic patients. The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 91%, 100% and 93 %, respectively. RT-dPCR is highly accurate method and suitable for detection of pharyngeal swab samples from COVID-19 suspected patients and patients under isolation and observation who may not be exhibiting clinical symptoms.