These files contain RNAseq dataset reported in "A mutation in RNA polymerase that imparts resistance to β-lactams by preventing dysregulation of amino acid and nucleotide metabolism" by Patel et al. In this study, we compared two RNAP mutations with opposite effects on β-lactam resistance in Bacillus subtilis. A substitution in β’ (rpoC G1122D) increased resistance, whereas one in β (rpoB H482Y) increased sensitivity. RNA sequencing uncovered a decrease in branched-chain amino acid (BCAA), methionine, and pyrimidine pathways specifically in the β-lactam-resistant strain. These same pathways were up-regulated by cefuroxime (CEF), a model β-lactam in wild-type, but not in the CEF-resistant rpoC mutant. Genetic derepression of BCAA or pyrimidine synthesis also increased CEF sensitivity. Thus, β-lactams can alter the transcriptome and thereby contribute to drug toxicity, and RNAP mutations can impede these deleterious changes. These findings provide a vivid example of how RNAP mutations, which commonly arise in response to diverse selection conditions, can alter the transcriptome resulting in a rewiring of cellular metabolism to enhance fitness.