The rpoB gene encodes the β subunit of bacterial RNA polymerase. rpoB is also found in plant chloroplasts where it forms the beta subunit of the plastid-encoded RNA polymerase (PEP). An inhibitor of transcription in bacteria, tagetitoxin, also inhibits PEP, showing that the complex found in plants is very similar to the homologous enzyme in bacteria. It codes for 1342 amino acids, making it the second-largest polypeptide in the bacterial cell. It is the site of mutations that confer resistance to the rifamycin antibacterial agents, such as rifampin. Mutations in rpoB that confer resistance to rifamycins do so by altering residues of the rifamycin binding site on RNA polymerase, thereby reducing rifamycin binding affinity for rifamycins
|DNA-directed RNA polymerase subunit beta|
|Chromosome||genomic: 4.18 - 4.19 Mb|
Some bacteria contain multiple copies of the 16S rRNA gene, which is commonly used as the molecular marker to study phylogeny. In these cases, the rpoB gene can be used to study microbial diversity.
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.
Physiological Effects of rpoB MutationsEdit
The regions of the rpoB gene which are susceptible to mutations are typically well conserved, indicating they are important for life. This makes it very likely that mutations within these regions have some effect on the overall fitness of the organism. These physiological changes can include a reduced rate of growth, increased sensitivity to increases or decreases in temperature, and alterations to the properties of RNA chain elongation and transcription termination. Such changes are not universal across all bacteria, though. A mutation in codon 450 of M. tuberculosis leads to a minor loss of fitness, while the corresponding mutation in S. aureus results in bacteria barely able to survive.
In Neisseria meningitidis rpoB mutations have been observed to increase expression of enzymes which are involved in metabolizing carbohydrates, as well as enzymes involved in the citric acid cycle and in transcription elongation. At the same time enzymes involved in ATP production, cell division, and lipid metabolism are all downregulated, or expressed at a lower than normal level.
In M. tuberculosis mutations in the rpoB gene can significantly upregulate polyketide synthase, potentially indicating increased production of phthiocerol dimycocerosate, a lipid produced by M. tuberculosis and implicated in virulence of the bacteria. Mutations also impact promoter binding, elongation, termination, and transcription-coupled repair processes in the RNA polymerase itself. Because of this, rpoB mutations were used to study transcription mechanisms before interest shifted to their ability to impart antibiotic resistance. Particular mutations can even result in strains of M. tuberculosis which grow better in the presence of rifampicin than they do when the antibiotic is not present.
In bacteria which are used to produce naturally occurring antibiotics such as erythromycin (Saccharopolyspora erythraea) and vancomycin (Amycolatopsis orientalis) certain rpoB mutations can increase the production of antibiotic by bacteria with those mutations.
- Börner T, Aleynikova AY, Zubo YO, Kusnetsov VV (September 2015). "Chloroplast RNA polymerases: Role in chloroplast biogenesis" (PDF). Biochimica et Biophysica Acta. 1847 (9): 761–9. doi:10.1016/j.bbabio.2015.02.004. PMID 25680513.
- Goldstein BP (September 2014). "Resistance to rifampicin: a review". The Journal of Antibiotics. 67 (9): 625–30. doi:10.1038/ja.2014.107. PMID 25118103.
- Floss HG, Yu TW (February 2005). "Rifamycin-mode of action, resistance, and biosynthesis". Chemical Reviews. 105 (2): 621–32. doi:10.1021/cr030112j. PMID 15700959.
- Campbell EA, Korzheva N, Mustaev A, Murakami K, Nair S, Goldfarb A, Darst SA (March 2001). "Structural mechanism for rifampicin inhibition of bacterial rna polymerase". Cell. 104 (6): 901–12. doi:10.1016/S0092-8674(01)00286-0. PMID 11290327.
- Feklistov A, Mekler V, Jiang Q, Westblade LF, Irschik H, Jansen R, Mustaev A, Darst SA, Ebright RH (September 2008). "Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center". Proceedings of the National Academy of Sciences of the United States of America. 105 (39): 14820–5. doi:10.1073/pnas.0802822105. PMC 2567451. PMID 18787125.
- Case RJ, Boucher Y, Dahllöf I, Holmström C, Doolittle WF, Kjelleberg S (January 2007). "Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies". Applied and Environmental Microbiology. 73 (1): 278–88. doi:10.1128/AEM.01177-06. PMC 1797146. PMID 17071787.
- Vos M, Quince C, Pijl AS, de Hollander M, Kowalchuk GA (2012). "A comparison of rpoB and 16S rRNA as markers in pyrosequencing studies of bacterial diversity". PLOS One. 7 (2): e30600. doi:10.1371/journal.pone.0030600. PMC 3280256. PMID 22355318.
- Alifano P, Palumbo C, Pasanisi D, Talà A (May 2015). "Rifampicin-resistance, rpoB polymorphism and RNA polymerase genetic engineering". Journal of Biotechnology. 202: 60–77. doi:10.1016/j.jbiotec.2014.11.024. PMID 25481100.
- Koch A, Mizrahi V, Warner DF (March 2014). "The impact of drug resistance on Mycobacterium tuberculosis physiology: what can we learn from rifampicin?". Emerging Microbes & Infections. 3 (3): e17. doi:10.1038/emi.2014.17. PMC 3975073. PMID 26038512.
- Jin DJ, Gross CA (July 1988). "Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance". Journal of Molecular Biology. 202 (1): 45–58. doi:10.1016/0022-2836(88)90517-7. PMID 3050121.
- Mokrousov I, Otten T, Vyshnevskiy B, Narvskaya O (July 2003). "Allele-specific rpoB PCR assays for detection of rifampin-resistant Mycobacterium tuberculosis in sputum smears". Antimicrobial Agents and Chemotherapy. 47 (7): 2231–5. doi:10.1128/AAC.47.7.2231-2235.2003. PMC 161874. PMID 12821473.
- Telenti A, Imboden P, Marchesi F, Lowrie D, Cole S, Colston MJ, Matter L, Schopfer K, Bodmer T (March 1993). "Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis". Lancet. 341 (8846): 647–50. doi:10.1016/0140-6736(93)90417-F. PMID 8095569.
- Wichelhaus TA, Schäfer V, Brade V, Böddinghaus B (November 1999). "Molecular characterization of rpoB mutations conferring cross-resistance to rifamycins on methicillin-resistant Staphylococcus aureus". Antimicrobial Agents and Chemotherapy. 43 (11): 2813–6. doi:10.1128/aac.43.11.2813. PMC 89569. PMID 10543773.