Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins.
Rifamycins, the clinically important antibiotics, target bacterial RNA polymerase (RNAP). A proposed mechanism in which rifamycins sterically block the extension of nascent RNA beyond three nucleotides does not alone explain why certain RNAP mutations confer resistance to some but not other rifamycins. Here we show that unlike rifampicin and rifapentin, and contradictory to the steric model, rifabutin inhibits formation of the first and second phosphodiester bonds. We report 2.5 A resolution structures of rifabutin and rifapentin complexed with the Thermus thermophilus RNAP holoenzyme. The structures reveal functionally important distinct interactions of antibiotics with the initiation sigma factor. Strikingly, both complexes lack the catalytic Mg2+ ion observed in the apo-holoenzyme, whereas an increase in Mg2+ concentration confers resistance to rifamycins. We propose that a rifamycin-induced signal is transmitted over approximately 19 A to the RNAP active site to slow down catalysis. Based on structural predictions, we designed enzyme substitutions that apparently interrupt this allosteric signal.
Artsimovitch
Irina
I
Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA.
Vassylyeva
Marina N
MN
Svetlov
Dmitri
D
Svetlov
Vladimir
V
Perederina
Anna
A
Igarashi
Noriyuki
N
Matsugaki
Naohiro
N
Wakatsuki
Soichi
S
Tahirov
Tahir H
TH
Vassylyev
Dmitry G
DG
D000494:Allosteric Regulation; D000900:Anti-Bacterial Agents / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000494:pharmacology*; D002384:Catalysis; D018360:Crystallography, X-Ray; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D015394:Molecular Structure; D011487:Protein Conformation; D012294:Rifamycins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000187:drug effects / Q000201:enzymology; D014157:Transcription Factors / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism
D000900:Anti-Bacterial Agents; D012294:Rifamycins; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium