By Levi Clancy for Student Reader on
dsDNA 40kb genome.
Complex icosohedral head & short tail.
More completely dominates cell than M13.
Replication cycle starts; within 6 minutes host cell protein synthesis shut off.
Viral proteins completely degrade host cell DNA.
Host nucleotides used for virion genome replication
Per cell, virion makes 200 copies of viral DNA within 15-20 post-infection.
T7 good model organism for gene expression....dsDNA....simply 55 gene 40kb genome....infect many viral DNAs per cell...isolate viral mutants
Pulse-Labeling of viral proteins in E. coli
Add T7 (MOI of 5pfu/cell) at t=0
Remove 5mL aliquots every 2min
Add 35S-methionine for 30 sec (this makes newly synthesized fluoresce)
Lyse cells in SDS
Subject to polyacrylimide gel electrophoresis
Prepare autoradiogram of gel
Steps to Identify Regulatory Mechanism for Gene Expression
There are several possible ways the virus can regulate gene expression.
Translational or transcriptional control
The experiment to determine which mechanism is used goes as follow:
Identify Class I proteins
Map location of each Class I gene by mapping RNA for each protein
Make mutants in each Class I gene
Analyze the phenotype of each mutant
To identify Class I proteins, proteins in T7-infected cells are pulse-labeled at various time post-infection. Next, the temperature-sensitive mutant experiment is performed. Temperature-sensitive mutants grow at the permissive temperature of 32Â°C; they do not grow at 39Â°C. Temperature-sensitive mutants in gene 1 do not express Class II and Class III genes at the non-permissive temperature. The methodology is as follows:
T7-infect cells are pulse-labeled at various times post-infection.
Profile of TS-infected cells at non-permissive temperature is compared to the profile of TS-infected cells at the permissive temperature.
Are TS1 mutants blocked at the level of transcription? protein profile at non-permissive temperature? Are ts1 mutants blocked at transcriptional level? test....infect cells with high MOI at nonpermissive, label with 3H-uridine...extract RNA and hybridize to cloned DNAs of each class of gene...
There is an experiment designed to test this idea:
Infect cells with high MOI at the nonpermissive temperature.
Label with 3H-uridine.
Extract RNA and hybridize to cloned DNAs of each class of gene.
|Temperature||Time||Class I||Class II||Class III|
TS1 mutants in gene 1 do not express Class II and Class III genes at the nonpermissive temperature. We design an experiment to see if there is a polymerase encoded in Gene 1 that is necessary to transcribe Class II and Class III genes. The experiment is as follows:
Gel Filtration: An extract of E. coli proteins is poured over a sizing column. Proteins go in and out of beads with different sized holes. Larger proteins elute first; smaller proteins elute later because they get trapped inside the beads. We find a 450kD cellular RNAP and the 98kD viral RNAP. the cellular RNAP has activity initially, but then the viral RNAP replaces it to transcribe the later genes. The conclusion: Gene 1 encodes a T7-specific RNAP responsible for transcribing Class II and Class III genes.
The temporal order of T7 gene expression is in the same order as the genes themselves, from left to right. There is a terminator for E. Coli RNAP before at the beginning of Class II and Class III genes. Class II and Class III promoters have a common DNA sequence different from Class I promoters. Class I promoters are transcribed by the E. Coli's RNAP. Class II and Class III promoters are transcribed by the T7-encoded RNAP, which recognizes its own promoter sequence.
Summary of T7 Experiments:
PAGE gel showed three waves of viral proteins.
Temperature sensitive mutants were developed in Class I genes.
The phenotype of gene I mutants was that there was no transcription of viral Class II and Class III genes at the non-permissive temperature.
Extracts from WT infected cells show a viral-specific RNAP eluting from an ion exchange column.
Exctracts from TS1 infected cells at the non-permissive temperature do not show this viral RNAP activity.
The conclusion is that gene 1 encodes a T7-specific RNAP responsible for transcribing Class II and Class III promoters.
Class II and Class III promoters have a common sequence different from Class I promoters, which are transcribed by the cell's RNAP.
This figure shows the activity of the cellular RNA polymerase and the activity of the T7 RNA polymerase as a function of column fractions. How do you think the activity of each of the RNA polymerases was measured? Viral RNAP activity replaces and is greater than cellular RNAP. Activity could be measured by labeling the promoters with different fluorescent genes. The degree of fluorescence would be proportional to RNAP activity.