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Explain the general facts about T7
it is a model phage (phage group) that was picked out from a commercial mixture called phage therapy treatments. Though it showed promise, when antibiotics appeared, phage therapy was dropped
Explain the virion.
Complex virus with short tail and short tail fibers; 60 nm diameter
starts off with a naked icosahedral
really ordered and intricate structures attached to the icosahedral wall; head and tail virus that physically injects contents of the virus inside its cell
Explain the genome
- really big virus with a T=7 (7x60)
- 56 open reading frames code for 59 proteins
has three classes of genes that are organized into blocks from left to right on the genome
Explain the organization of the classes of genes
they are clustered both by function and by the timing of their expression during the life cycle of the phage
Class I: needed for expression of the remainder of phage genes and to shut down host cell functions
Class II: occupy the next one-third of the genome. Most are concerned with phage DNA replication
Class III: code for phage structural proteins and proteins needed for DNA maturation and packaging
Class I in detail
- first 6-8 minutes of infection
- control expression of phage genes and host shutoff
middle genes that control phage DNA replication
- structural proteins
- proteins for DNA maturation and packaging
Explain entry of virus into bacteria
Looks and behaves as a syringe
The tail fibers bind to the outer membrane of bacteria. A conformational change occurs and a core proteins form a channel through the outer membrane, wall, and cytoplasmic membrane. 850 bp of viral DNA is transported into the cell
Virus binds to lipopolysaccharide on the outer membrane
Another conformational change allows transportation of the first 850 bps of the DNA, which exposes three promoters recognized by E. coli RNA plymerase. At this point, transcription occurs, pulling the DNA into the cytoplasm.
Therefore, RNA polymerase transfers the DNA from the capsid into the cell while making mRNA
Explain the progression of transcription.
The early genes need to be transcribed first to have a successful infection
The virus makes proteins that inhibit restriction endonucleases.
They also make viral RN polymerase, which drives the rest of transcription of the different genes.
Promoters recognized by the phage RNA polymerase are recognized; and, new transcripts and proteins are made
What do Class II genes code for?
enzymes involved in T7 DNA replication, such as DNA ligase, SSBP, endonucleases, lysozyme, primase and helicases, DNA polymerase, and exonucleases (degrade host cell DNA)
What is the function of Class III genes?
- portal protein (connector for tail)
- scaffolding protein
- capsid protein
- tail protein
- core proteins
AKA: structural components
What is the regulation of Class III gene expression.
delayed entry and promoter strength
transcription-dependent DNA entry delays transcription
Class III promoters ahve a higher affinity for T7 RNA polymerase
Explain genetic engineering wth T7.
By putting a T7 promoter in front of a gene of interest, one can make a variety of expression vectors, which allow regulated expression and high yields of genes of interest
Explain in detail transcription of class II and III genes?
requires a novel T7- coded RNA polymerase
- Class 1 gene 1.0 is a viral RNA polymerase that transcribes Class II and III genes
- Gene 0.3 protects viral DNA from degradation
- Gene 0.7 shuts off cellular transcription
Specificity of T7 polymerase
helps to regulate gene expression