Transcription inhibition by the depsipeptide antibiotic salinamide A.
We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center 'bridge-helix cap' comprising the 'bridge-helix N-terminal hinge', 'F-loop', and 'link region'. We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.DOI: http://dx.doi.org/10.7554/eLife.02451.001.
Waksman Institute, Rutgers University, Piscataway, United States.
David
D
Degen
Waksman Institute, Rutgers University, Piscataway, United States.
Yu
Y
Feng
Waksman Institute, Rutgers University, Piscataway, United States.
Yu
Y
Zhang
Waksman Institute, Rutgers University, Piscataway, United States.
Katherine Y
KY
Ebright
Waksman Institute, Rutgers University, Piscataway, United States.
Yon W
YW
Ebright
Waksman Institute, Rutgers University, Piscataway, United States.
Matthew
M
Gigliotti
Waksman Institute, Rutgers University, Piscataway, United States.
Hanif
H
Vahedian-Movahed
Waksman Institute, Rutgers University, Piscataway, United States.
Sukhendu
S
Mandal
Center for Biodefense, New Jersey Medical School, Rutgers University, Newark, United States.
Meliza
M
Talaue
Center for Biodefense, New Jersey Medical School, Rutgers University, Newark, United States.
Nancy
N
Connell
Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, United States.
Eddy
E
Arnold
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, United States.
William
W
Fenical
Waksman Institute, Rutgers University, Piscataway, United States ebright@waksman.rutgers.edu.
Richard H
RH
Ebright
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001966:Bromine; D018360:Crystallography, X-Ray; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D047630:Depsipeptides / Q000737:chemistry / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects*
D000900:Anti-Bacterial Agents; D047630:Depsipeptides; D004791:Enzyme Inhibitors; D009711:Nucleotides; D021122:Protein Subunits; C529014:salinamide A; D012321:DNA-Directed RNA Polymerases; D001966:Bromine
RNA polymerase; antibiotic; bridge helix; bridge-helix cap; inhibitor; transcription