Rifampicin (Rif) is one of the most potent and broad spectrum antibiotics against bacterial pathogens and is a key component of anti-tuberculosis therapy, stemming from its inhibition of the bacterial RNA polymerase (RNAP). We determined the crystal structure of Thermus aquaticus core RNAP complexed with Rif. The inhibitor binds in a pocket of the RNAP beta subunit deep within the DNA/RNA channel, but more than 12 A away from the active site. The structure, combined with biochemical results, explains the effects of Rif on RNAP function and indicates that the inhibitor acts by directly blocking the path of the elongating RNA when the transcript becomes 2 to 3 nt in length.

The path of the nucleic acids through a transcription elongation complex was tracked by mapping cross-links between bacterial RNA polymerase (RNAP) and transcript RNA or template DNA onto the x-ray crystal structure. In the resulting model, the downstream duplex DNA is nestled in a trough formed by the beta' subunit and enclosed on top by the beta subunit. In the RNAP channel, the RNA/DNA hybrid extends from the enzyme active site, along a region of the beta subunit harboring rifampicin resistance mutations, to the beta' subunit "rudder." The single-stranded RNA is then extruded through another channel formed by the beta-subunit flap domain. The model provides insight into the functional properties of the transcription complex.

Cold Spring Harbor Symposia on Quantitative Biology, Volume LXIII. © 1998 Cold Spring Harbor Laboratory Press 0-87969-550-1/98. Cold Spring Harbor Laboratory Press on October 24, 2012 -Published by symposium.cshlp.org Downloaded from 340 KORZHEVA ET AL.Figure 3. Salt stability of TEC in the Minimal Product System. (A) Route to the TEC with short RNA; (B) time course of the decay of TECs generated by pyrophosphorolysis of TEC carried 11-nucleotide RNA; (C) graphic representation of the data from B. Each of the 8-, 9-, 10-, and 11-nucleotide products displayed stability within the limits represented by vertical bars. (D) Stability of TEC produced by RNase T1 cleavage of 26 TECs and subsequent pyrophosphorolysis. (Lane 1) Original 26-nucleotide TEC; (lane 2) the 16-nucleotide TEC is the RNase T1 cleavage product of the 26-nucleotide TEC; (lanes 3-8) time course for the decay of TECs obtained by pyrophosphorolysis in lane 2.

contributed equally to this work Analysis of multisubunit RNA polymerase (RNAP) structures revealed several elements that may constitute the enzyme's functional sites. One such element, the`rudder', is formed by an evolutionarily conserved segment of the largest subunit of RNAP and contacts the nascent RNA at the upstream edge of the RNA±DNA hybrid, where the DNA template strand separates from the RNA transcript and re-anneals with the non-template strand. Thus, the rudder could (i) maintain the correct length of the RNA±DNA hybrid; (ii) stabilize the nascent RNA in the complex; and (iii) promote or maintain localized DNA melting at the upstream edge of the bubble. We generated a recombinant RNAP mutant that lacked the rudder and studied its properties in vitro. Our results demonstrate that the rudder is not required for establishment of the upstream boundary of the transcription bubble during promoter complex formation, nor is it required for separation of the nascent RNA from the DNA template strand or transcription termination. Our results suggest that the rudder makes critical contributions to elongation complex stability through direct interactions with the nascent RNA.

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