Faculty Bibliography

A mutation in the conserved segment of the rpoC gene, which codes for the largest RNA polymerase (RNAP) subunit, beta', was found to make Escherichia coli cells resistant to microcin J25 (MccJ25), a bactericidal 21-amino acid peptide active against Gram-negative bacteria (Delgado, M. A., Rintoul, M. R., Farias, R. N., and Salomon, R. A. (2001) J. Bacteriol. 183, 4543-4550). Here, we report that mutant RNAP prepared from MccJ25-resistant cells, but not the wild-type RNAP, is resistant to MccJ25 in vitro, thus establishing that RNAP is a true cellular target of MccJ25. We also report the isolation of additional rpoC mutations that lead to MccJ25 resistance in vivo and in vitro. The new mutations affect beta' amino acids in evolutionarily conserved segments G, G', and F and are exposed into the RNAP secondary channel, a narrow opening that connects the enzyme surface with the catalytic center. We also report that previously known rpoB (RNAP beta subunit) mutations that lead to streptolydigin resistance cause resistance to MccJ25. We hypothesize that MccJ25 inhibits transcription by binding in RNAP secondary channel and blocking substrate access to the catalytic center.

Each elementary step of transcription involves translocation of the 3' terminus of RNA in the RNA polymerase active center, followed by the entry of a nucleoside triphosphate. The structural basis of these transitions was studied using RNA-protein crosslinks. The contacts were mapped and projected onto the crystal structure, in which the "F bridge" helix in the beta' subunit is either bent or relaxed. Bending/relaxation of the F bridge correlates with lateral movements of the RNA 3' terminus. The bent conformation is sterically incompatable with the occupancy of the nucleotide site, suggesting that the switch regulates both the entry of substrates and the translocation of the transcript. The switch occurs as part of a cooperative transition of a larger structural domain that consists of the F helix and the supporting G loop.