Functional analysis of mutations in the transcription terminator T₁ that suppress two dnaG alleles in Escherichia coli

Abstract

The mutations dnaG2903 and parB are both temperature-sensitive conditional lethal alleles of the Escherichia coli dnaG gene, which encodes the protein primase. The lesions are located in the 3′ end of the gene, 9 basepairs apart, and both cause Glu-to-Lys substitutions in the carboxy terminus of primase. Previously, it was shown that dnaG2903 can be suppressed by point mutations in the rho-independent transcription terminator T₁, which is located just upstream of dnaG in the rpsU-dnaG-rpoD macromolecular synthesis operon. We report here that parB can also be suppressed by point mutations in T₁, demonstrating that parB can be suppressed in the same manner as dnaG2903. We also identified additional suppressors of dnaG2903 that are point mutations in T₁, suggesting that defective transcription termination leading to overexpression of dnaG2903 and parB suppresses the temperature-sensitive phenotype of strains harboring these mutations. Utilizing two mutant rpoB alleles whose transcription termination phenotypes at rho-independent terminators have been previously characterized, we demonstrate that defective transcription termination leading to the overexpression of dnaG does indeed suppress dnaG2903 and parB. The point mutations in T₁ identified in this study were analyzed for their effects on termination efficiency at T₁. Our results indicate that the thermodynamic stability of the hairpin structures may not be the sole determinant of termination efficiency in vivo.