SummaryThis work provides evidence that, during transcription, the mutability (propensity to mutate) of a base in a DNA secondary structure depends both on the stability of the structure and on the extent to which the base is unpaired. Zuker ' s DNA folding computer program reveals the most probable stem-loop structures (SLSs) and negative energies of folding (-∆ ∆ ∆ ∆ G) for any given nucleotide sequence. We developed an interfac-
This article presents evidence that starvation for leucine in an Escherichia coli auxotroph triggers metabolic activities that specifically target the leu operon for derepression, increased rates of transcription, and mutation. Derepression of the leu operon was a prerequisite for its activation by the signal nucleotide, guanosine tetraphosphate, which accumulates in response to nutritional stress (the stringent response). A quantitative correlation was established between leuB mRNA abundance and leuB ؊ reversion rates. To further demonstrate that derepression increased mutation rates, the chromosomal leu operon was placed under the control of the inducible tac promoter. When the leu operon was induced by isopropyl-D-thiogalactoside, both leuB mRNA abundance and leuB ؊ reversion rates increased. These investigations suggest that guanosine tetraphosphate may contribute as much as attenuation in regulating leu operon expression and that higher rates of mutation are specifically associated with the derepressed leu operon.
Escherichia coli auxotrophs of leuB and argH were examined to determine if higher rates of transcription in derepressed genes were correlated with increased reversion rates. Rates of leuB and argH mRNA synthesis were determined using half-lives and concentrations, during exponential growth and at several time points during 30 min of amino acid starvation. Changes in mRNA concentration were primarily due to increased mRNA synthesis and not to increased stability. Four strains of E. coli amino acid auxotrophs, isogenic except for relA and argR, were examined. In both the leuB and argH genes, rates of transcription and mutation were compared. In general, strains able to activate transcription with guanosine tetraphosphate (ppGpp) had higher rates of mRNA synthesis and mutation than those lacking ppGpp (relA2 mutants). argR knockout strains were constructed in relA + and relA mutant strains, and rates of both argH reversion and mRNA synthesis were significantly higher in the argR knockouts than in the regulated strains. A statistically significant linear correlation between increased rates of transcription and mutation was found for data from both genes. In general, changes in mRNA half-lives were less than threefold, whereas changes in rates of mRNA synthesis were often two orders of magnitude. The results suggest that specific starvation conditions target the biosynthetic genes for derepression and increased rates of transcription and mutation.
SummaryFour mutations resulting in opal stop codons were individually engineered into a plasmid-borne chloramphenicol-resistance ( cat ) gene driven by the lac promoter. These four mutations were located at different sites in secondary structures. The mutations were analysed with the computer program mfg , which predicted their relative reversion frequencies. Reversion frequencies determined experimentally correlated with the mutability of the bases as predicted by mfg . To examine the effect of increased transcription on reversion frequencies, the lac promoter was replaced with the stronger tac promoter, which resulted in 12-to 30-fold increases in reversion rates. The effect of increased and decreased supercoiling was also investigated. The cat mutants had higher reversion rates in a topA mutant strain with increased negative supercoiling compared with wild-type levels, and the cat reversion rates were lower in a topA gyrB mutant strain with decreased negative supercoiling, as predicted.
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