BACKGROUND: During transcription, the nontranscribed DNA strand becomes 
single-stranded DNA (ssDNA), which can form secondary structures. Unpaired bases 
in the ssDNA are less protected from mutagens and hence experience more 
mutations than do paired bases. These mutations are called 
transcription-associated mutations. Transcription-associated mutagenesis is 
increased under stress and depends on the DNA sequence. Therefore, selection 
might significantly influence protein-coding sequences in terms of the 
transcription-associated mutability per transcription event under stress to 
improve the survival of Escherichia coli.
METHODOLOGY/PRINCIPAL FINDINGS: The mutability index (MI) was developed by 
Wright et al. to estimate the relative transcription-associated mutability of 
bases per transcription event. Using the most stable fold of each ssDNA that 
have an average length n, MI was defined as (the number of folds in which the 
base is unpaired)/nx(highest -DeltaG of all n folds in which the base is 
unpaired), where DeltaG is the free energy. The MI values show a significant 
correlation with mutation data under stress but not with spontaneous mutations 
in E. coli. Protein sequence diversity is preferred under stress but not under 
favorable conditions. Therefore, we evaluated the selection pressure on MI in 
terms of the protein sequence diversity for all the protein-coding sequences in 
E. coli. The distributions of the MI values were lower at bases that could be 
substituted with each of the other three bases without affecting the amino acid 
sequence than at bases that could not be so substituted. Start codons had lower 
distributions of MI values than did nonstart codons.
CONCLUSIONS/SIGNIFICANCE: Our results suggest that the majority of 
protein-coding sequences have evolved to promote protein sequence diversity and 
to reduce gene knockout under stress. Consequently, transcription-associated 
mutagenesis increases protein sequence diversity more effectively than does 
random mutagenesis under stress. Nonrandom transcription-associated mutagenesis 
under stress should improve the survival of E. coli.