In a bacterium without the proper mutation(s) in rpoB rifampicin binds to a site near the fork in the β subunit and prevents the polymerase from transcribing more than two or three base pairs of any RNA sequence and stopping production of proteins within the cell. Bacteria with mutations in the proper loci along the rpoB gene are resistant to this effect.
Initial studies were done by Jin and Gross to generate rpoB mutations in E. coli that conferred resistance to rifampicin. Three clusters of mutations were identified, cluster I at codons 507-533, cluster II at codons 563-572, and cluster III at codon 687. The majority of these mutations are located within an 81 base pair(bp) region in cluster I dubbed the "Rifampicin Resistance Determining Region (RRDR)". This resistance is typically associated with a mutation wherein a base in the DNA is substituted for another one and the new sequence codes for an amino acid with a large side chain that inhibits the rifampicin molecules from binding to the polymerase.
There are additional mutations which can occur in the β subunit of the polymerase which are located away from the rifampicin binding site that can also result in mild resistance. Potentially indicating that the shape of these areas may affect the formation of the rifampicin binding site.
Nucleic acid probes can detect mutations in rpoB that confer rifampicin resistance.  For Mycobacterium tuberculosis, the rifamycin-resistant mutations most commonly encountered involve codons 516, 526, and 531 (numbered, by convention, as in Escherichia coli rpoB). These mutations result in high rifampicin resistance with a relatively low loss of fitness.  For Staphylococcus aureus, the rifamycin-resistant mutation most commonly encountered involves codon 526.
In addition to imparting resistance to rifampicin, certain rpoB mutations have been identified in 70% of Vancomycin Intermediate S. aureus (VISA) strains.