Viral genome packaging terminase cleaves DNA using the canonical RuvC-like two-metal catalysis mechanism

Bacteriophages and large dsDNA viruses encode sophisticated machinery to translocate their DNA into a preformed empty capsid. An essential part of this machine, the large terminase protein, processes viral DNA into constituent units utilizing its nuclease activity. Crystal structures of the large terminase nuclease from the thermophilic bacteriophage G20c show that it is most similar to the RuvC family of the RNase H-like endonucleases. Like RuvC proteins, the nuclease requires either Mn2+, Mg2+ or Co2+ ions for activity, but is inactive with Zn2+ and Ca2+. High resolution crystal structures of complexes with different metals reveal that in the absence of DNA, only one catalytic metal ion is accommodated in the active site. Binding of the second metal ion may be facilitated by conformational variability, which enables the two catalytic aspartic acids to be brought closer to each other. Structural comparison indicates that in common with the RuvC family, the location of the two catalytic metals differs from other members of the RNase H family. In contrast to a recently proposed mechanism, the available data do not support binding of the two metals at an ultra-short interatomic distance. Thus we postulate that viral terminases cleave DNA by the canonical RuvC-like mechanism

Cryo-EM structure and in vitro DNA packaging of a thermophilic virus with supersized T=7 capsids

Double-stranded DNA viruses, including bacteriophages and herpesviruses, package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus Both the unexpanded procapsid and the expanded mature capsid can package DNA in the presence of packaging ATPase over the 20 °C to 70 °C temperature range, with optimum activity at 50 °C to 65 °C. Cryo-EM reconstructions for the mature and immature capsids at 3.7-Å and 4.4-Å resolution, respectively, reveal conformational changes during capsid expansion. Capsomer interactions in the expanded capsid are reinforced by formation of intersubunit β-sheets with N-terminal segments of auxiliary protein trimers. Unexpectedly, the capsid has T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of known T=7 phages, in which the DNA is compacted to near-crystalline density. Our data explain this anomaly, showing how the canonical HK97 fold has adapted to double the volume of the capsid, while maintaining its structural integrity. Reconstructions of the procapsid and the expanded capsid defined the structure of the single vertex containing the portal protein. Together with a 1.95-Å resolution crystal structure of the portal protein and DNA packaging assays, these reconstructions indicate that capsid expansion affects the conformation of the portal protein, while still allowing DNA to be packaged. These observations suggest a mechanism by which structural events inside the capsid can be communicated to the outside

Novel Escherichia coli RNA Polymerase Binding Protein Encoded by Bacteriophage T5

The Escherichia coli bacteriophage T5 has three temporal classes of genes (pre-early, early, and late). All three classes are transcribed by host RNA polymerase (RNAP) containing the σ70 promoter specificity subunit. Molecular mechanisms responsible for the switching of viral transcription from one class to another remain unknown. Here, we find the product of T5 gene 026 (gpT5.026) in RNAP preparations purified from T5-infected cells and demonstrate in vitro its tight binding to E. coli RNAP. While proteins homologous to gpT5.026 are encoded by all T5-related phages, no similarities to proteins with known functions can be detected. GpT5.026 binds to two regions of the RNAP β subunit and moderately inhibits RNAP interaction with the discriminator region of σ70-dependent promoters. A T5 mutant with disrupted gene 026 is viable, but the host cell lysis phase is prolongated and fewer virus particles are produced. During the mutant phage infection, the number of early transcripts increases, whereas the number of late transcripts decreases. We propose that gpT5.026 is part of the regulatory cascade that orchestrates a switch from early to late bacteriophage T5 transcription

Temporal regulation of gene expression of the Escherichia coli bacteriophage phiEco32

Escherichia coli phage phiEco32 encodes two proteins that bind to host RNA polymerase (RNAP): gp79, a novel protein, and gp36, a distant homolog of σ(70) family proteins. Here, we investigated the temporal pattern of phiEco32 and host gene expression during infection. Host transcription shutoff and three distinct bacteriophage temporal gene classes (early, middle, and late) were revealed. A combination of bioinformatic and biochemical approaches allowed identification of phage promoters recognized by a host RNAP holoenzyme containing the σ(70) factor. These promoters are located upstream of early phage genes. A combination of macroarray data, primer extension, and in vitro transcription analyses allowed identification of six promoters recognized by an RNAP holoenzyme containing gp36. These promoters are characterized by a single-consensus element tAATGTAtA and are located upstream of the middle and late phage genes. Curiously, gp79, an inhibitor of host and early phage transcription by σ(70) holoenzyme, activated transcription by the gp36 holoenzyme in vitro

Components of positive impact of exposure on university physical culture and sports on students' physical activity

This article presents an analysis of activities performed by teachers and administrative personnel of higher education institutions to create and make the best use of the physical culture and sports environment in higher education establishments. It was revealed that this environment should rest on the following structural components: rating scales for athletic performance evaluation of students and contemporary methods for teaching physical education lessons at universities. The efficiency of using rating score systems to measure the athletic performance of students was confirmed by a significant increase (P<0.01) in the number of young people who participate in PE and sports on a regular basis and who take part in competitions of various levels. The application of cardio and strength training programs (HOT IRON) in physical education lessons has proved effective for students as well. The students in the experimental group significantly improved their muscle power (P<0.01) and overall endurance (0.05). These programs can also be applied for effective body shaping and obesity prevention

Components of positive impact of exposure on university physical culture and sports on students' physical activity

This article presents an analysis of activities performed by teachers and administrative personnel of higher education institutions to create and make the best use of the physical culture and sports environment in higher education establishments. It was revealed that this environment should rest on the following structural components: rating scales for athletic performance evaluation of students and contemporary methods for teaching physical education lessons at universities. The efficiency of using rating score systems to measure the athletic performance of students was confirmed by a significant increase (P<0.01) in the number of young people who participate in PE and sports on a regular basis and who take part in competitions of various levels. The application of cardio and strength training programs (HOT IRON) in physical education lessons has proved effective for students as well. The students in the experimental group significantly improved their muscle power (P<0.01) and overall endurance (0.05). These programs can also be applied for effective body shaping and obesity prevention