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Isolation of transcriptionally active mutants of T7 RNA polymerase that do not support phage growth
1Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
Abstract:
Mutants of bacteriophage T7 RNA polymerase defective in functions other than transcription were sought by random chemical mutagenesis of the cloned gene and selection for inability to support the growth of a T7 mutant whose growth is dependent on T7 RNA polymerase supplied by the host cell. About half of the mutant clones appeared unable to make full-length T7 RNA polymerase, many of them producing a truncated protein. Among 116 mutants expressing full-length protein, two-thirds were severely impaired in transcription, but a surprisingly high one-third were able to direct significant transcription in vivo. Both types of mutation were distributed across much of the gene, as determined by a rapid genetic mapping procedure that allows the lethal mutation in each clone to be localized. One mutation (isolated twice) allowed normal gene expression but prevented the formation of mature ends of T7 DNA from concatemers, which normally happens during packaging into phage particles. Thirty-seven of the mutations appeared to increase the sensitivity of the polymerase to inhibition by T7 lysozyme; all were suppressed by mutations in the lysozyme gene, including one suppressor constructed to retain full amidase activity but to be unable to bind T7 RNA polymerase. The two lysozyme-hypersensitive polymerase mutants analyzed in detail showed premature cessation of transcription during infection. Early proteins and those late proteins specified by genes as far right in T7 DNA as genes 8-9 appeared to be produced normally, but expression of genes farther to the right was strongly depressed. DNA replication was depressed about 50% in one of these mutants and 90% in the other, even though the T7 replication proteins were made in normal amounts at the normal time.
Insights
Researchers identified mutations in bacteriophage T7 RNA polymerase affecting its function beyond transcription. Some mutants showed impaired transcription, while others maintained significant activity, revealing new insights into polymerase function and regulation.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage T7 RNA polymerase is essential for viral gene expression.
- Understanding its functions beyond basic transcription is crucial for viral replication and host interactions.
- Previous studies have focused on core transcriptional activity, leaving other roles less explored.
Purpose of the Study:
- To identify and characterize bacteriophage T7 RNA polymerase mutants with defects in functions other than transcription.
- To investigate the roles of T7 RNA polymerase in viral DNA packaging and gene expression regulation.
- To explore the interaction between T7 RNA polymerase and T7 lysozyme.
Main Methods:
- Random chemical mutagenesis of the cloned T7 RNA polymerase gene.
- Selection of mutants unable to support the growth of a T7 host-dependent mutant.
- Genetic mapping to localize mutations.
- Analysis of protein truncation, transcriptional activity, DNA packaging, and lysozyme sensitivity.
Main Results:
- Approximately half of the mutants produced truncated T7 RNA polymerase.
- One-third of full-length mutants retained significant in vivo transcription.
- One mutation affected T7 DNA end formation during packaging.
- 37 mutations increased sensitivity to T7 lysozyme, impacting late gene expression and DNA replication.
Conclusions:
- T7 RNA polymerase possesses functions beyond transcription, including roles in DNA packaging and gene expression regulation.
- Lysozyme sensitivity is a key feature of some T7 RNA polymerase mutants, affecting downstream viral processes.
- Mutational analysis provides a powerful tool to dissect complex polymerase functions.