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The putative small terminase from the thermophilic dsDNA bacteriophage G20C is a nine-subunit oligomer.

ABSTRACT: The assembly of double-stranded DNA bacteriophages is dependent on a small terminase protein that normally plays two important roles. Firstly, the small terminase protein specifically recognizes viral DNA and recruits the large terminase protein, which makes the initial cut in the dsDNA. Secondly, once the complex of the small terminase, the large terminase and the DNA has docked to the portal protein, and DNA translocation into a preformed empty procapsid has begun, the small terminase modulates the ATPase activity of the large terminase. Here, the putative small terminase protein from the thermostable bacteriophage G20C, which infects the Gram-negative eubacterium Thermus thermophilus, has been produced, purified and crystallized. Size-exclusion chromatography-multi-angle laser light scattering data indicate that the protein forms oligomers containing nine subunits. Crystals diffracting to 2.8 Å resolution have been obtained. These belonged to space group P2?2?2?, with unit-cell parameters a = 94.31, b = 125.6, c = 162.8 Å. The self-rotation function and Matthews coefficient calculations are consistent with the presence of a nine-subunit oligomer in the asymmetric unit.

SUBMITTER: Loredo-Varela J

PROVIDER: S-EPMC3729163 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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The putative small terminase from the thermophilic dsDNA bacteriophage G20C is a nine-subunit oligomer.

Loredo-Varela Juan J Chechik Maria M Levdikov Vladimir M VM Abd-El-Aziz Ahmad A Minakhin Leonid L Severinov Konstantin K Smits Callum C Antson Alfred A AA

Acta crystallographica. Section F, Structural biology and crystallization communications 20130727 Pt 8

The assembly of double-stranded DNA bacteriophages is dependent on a small terminase protein that normally plays two important roles. Firstly, the small terminase protein specifically recognizes viral DNA and recruits the large terminase protein, which makes the initial cut in the dsDNA. Secondly, once the complex of the small terminase, the large terminase and the DNA has docked to the portal protein, and DNA translocation into a preformed empty procapsid has begun, the small terminase modulate ...[more]

PMID: 23908032

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Project description:To investigate the transcriptional profile of GVE2 genes, the viral genes were identified by DNA microarray with Cy5- or Cy3-dUTP-labeled cDNAs prepared from uninfected and GVE2-infected Geobacillus sp. E263 at 4 h p.i.. After hybridization with the Cy3-dUTP-labeled cDNAs from GVE2-infected Geobacillus sp. E263 at 4 h p.i., Cy5-dUTP-labeled cDNAs from uninfected Geobacillus sp. E263 as well as Cy3- dUTP-labeled yeast cDNAs and Hex DNA, many spots produced positive signals significantly above the background, while no signal appeared for the Cy5-dUTP-labeled cDNAs from uninfected Geobacillus sp. E263., indicating that the positive signals represented the GVE2 gene transcripts detectable by DNA microarray. The DNA fragments, detected to be positive in the reverse transcripts at 4 h p.i., contained 74.2% of the presumptive GVE2 ORFs. Keywords: Transcriptional profile of thermophilic bacteriophage at 4 h p.i. A DNA microarray containing 82 DNA fragments of the viral genome was constructed following a PCR-based microarray method. Briefly, specific primer sets were designed to amplify approximately 500-bp fragment each using viral genome as template. All PCR products showing a single band of the appropriate size by gel electrophoresis were purified, and reconstituted in TE buffer at a final concentration of about 500 Î¼g/ml for spotting in triplicates onto the silylated-glass slides (CEL Associates, Inc. USA) using a microarrayer (Smart Arrayer 48, CapitalBio). Eight DNA fragments from yeast genome and a randomly synthesized DNA fragment (Hex) were included as exogenous positive controls to normalize the microarry date. Distilled water was used as negative controls. Total RNAs were isolated from the uninfected and phage-infected Geobacillus sp. E263 cells at 4 h postinfection. The cDNAs from uninfected Geobacillus sp. E263 were labeled with Cy5 and the cDNAs from phage-infected Geobacillus sp. E263 labeled with Cy3. At the same time, the cDNAs from yeast and the Hex DNA were labeled with Cy3. The Cy5- or Cy3-dUTP-labeled cDNAs were resuspended in hybridization solution and hybridized with the microarrays for 16 to 18 h at 42Â°C. Then the microarrays were rinsed several times following the standard method. Following the washing steps, the microarrays were dried by low-speed centrifugation (500 g for 5 min), and immediately scanned using a GenePix 4000B array scanner (Axon Instruments, Inc.). Images obtained from scanning were analyzed by GenePix Pro 4.0 array analysis software (Axon Instruments, Inc.)

Dataset Information

The putative small terminase from the thermophilic dsDNA bacteriophage G20C is a nine-subunit oligomer.

Publications

The putative small terminase from the thermophilic dsDNA bacteriophage G20C is a nine-subunit oligomer.

Similar Datasets

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