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T7 protein synthesis in F' episome-containing cells: assignment of specific proteins to three translational groups
Abstract:
Synthesis of many T7 proteins is prevented in F' episome-containing cells. In order to quantitate the degree of inhibition, we measured the activity of several T7 proteins in extracts prepared from T7-infected F(-) and F' cells and cells containing F factors mutant in phage inhibition [F'(PIF(-)2A) and F'(PIF(-)2A,2B)]. In addition, we were able to assign specific T7 proteins to the three translational units previously defined by polyacrylamide gel analysis of T7 proteins made in F(-) and episome-containing cells. After T7 infection, the presence of the wild-type F' (PIF(+)) episome led to greater than 90% inhibition of T7 DNA polymerase (product of gene 5), T7 lysozyme (gene 3.5), and gene 10 capsid protein synthesis. Nearly normal amounts of T7 RNA polymerase (gene 1) were made in these cells. T7 infection of cells containing the mutant F' (PIF(-)2A) episome led to normal synthesis of T7 RNA polymerase and T7 DNA polymerase; T7 lysozyme was synthesized at 30% of the maximal level in these cells; T7 gene 10 capsid protein synthesis was inhibited by 90%, and T7 DNA synthesis was arrested in these cells. T7 infection of cells containing the mutant F' (PIF(-)2A,2B) episome led to synthesis of normal levels of the enzymes assayed.
Insights
The F' episome significantly inhibits T7 phage protein synthesis, particularly T7 DNA polymerase and capsid proteins. Mutant F' episomes partially restore synthesis, revealing specific T7 protein regulation mechanisms.
Area of Science:
- Molecular Biology
- Virology
- Bacteriology
Background:
- Bacteriophage T7 protein synthesis is known to be inhibited by the presence of F' episomes in bacterial cells.
- Understanding the specific mechanisms and extent of this inhibition is crucial for studying phage-host interactions.
Purpose of the Study:
- To quantify the inhibition of various T7 proteins during infection of F' episome-containing cells.
- To assign specific T7 proteins to defined translational units.
- To investigate the role of F' episome mutations in modulating T7 protein synthesis.
Main Methods:
- T7 phage infection of bacterial cells with wild-type and mutant F' episomes.
- Measurement of T7 protein activity (T7 DNA polymerase, T7 lysozyme, gene 10 capsid protein, T7 RNA polymerase) in cell extracts.
- Polyacrylamide gel analysis of T7 proteins.
Main Results:
- Wild-type F' episomes caused >90% inhibition of T7 DNA polymerase, T7 lysozyme, and gene 10 capsid protein synthesis, while T7 RNA polymerase synthesis remained normal.
- Mutant F'(PIF(-)2A) episomes allowed normal T7 RNA polymerase and T7 DNA polymerase synthesis, but inhibited T7 lysozyme (to 30%) and gene 10 capsid protein (by 90%), arresting T7 DNA synthesis.
- Mutant F'(PIF(-)2A,2B) episomes resulted in normal synthesis of all assayed enzymes.
Conclusions:
- The F' episome differentially regulates T7 protein synthesis, with specific translational units being sensitive to inhibition.
- Mutations in the F' episome's phage inhibition factors (PIF) can restore the synthesis of specific T7 proteins.
- This study elucidates the genetic basis of F' episome-mediated inhibition of T7 phage gene expression.