Isolation of transcriptionally active mutants of T7 RNA polymerase that do not support phage growth.
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.
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Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
Studier
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D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004252:DNA Mutational Analysis; D004261:DNA Replication / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005804:Genes, Lethal / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000494:pharmacology; D011995:Recombination, Genetic; D013489:Suppression, Genetic; D014158:Transcription, Genetic / Q000502:physiology*; D014764:Viral Proteins; D014779:Virus Replication / Q000235:genetics*
D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D009238:N-Acetylmuramoyl-L-alanine Amidase