TY - JOUR
T1 - Single-molecule studies reveal the off-pathway early paused state intermediates as a target of streptolydigin inhibition of RNA polymerase and its dramatic enhancement by Gre factors
AU - Arseniev, Anatolii
AU - Panfilov, Mikhail
AU - Pobegalov, Georgii
AU - Potyseva, Alina
AU - Pavlinova, Polina
AU - Yakunina, Maria
AU - Lee, Jookyung
AU - Borukhov, Sergei
AU - Severinov, Konstantin
AU - Khodorkovskii, Mikhail
N1 - Publisher Copyright: © The Author(s) 2024.
PY - 2025/1/13
Y1 - 2025/1/13
N2 - Antibiotic streptolydigin (Stl) inhibits bacterial transcription by blocking the trigger loop folding in the active center of RNA polymerase (RNAP), which is essential for catalysis. We use acoustic force spectroscopy to characterize the dynamics of transcription elongation in ternary elongation complexes (ECs) of RNAP in the presence of Stl at a single-molecule level. We found that Stl induces long-lived stochastic pauses while the instantaneous velocity of transcription between the pauses is unaffected. Stl enhances the short-lived pauses associated with an off-pathway early paused state intermediates of the RNAP nucleotide addition cycle. Unexpectedly, we found that transcript cleavage factors GreA and GreB, which were thought to be Stl competitors, do not alleviate the Stl-induced pausing; instead, they synergistically increase transcription inhibition by Stl. This is the first known instance of a transcriptional factor enhancing antibiotic activity. We propose a structural model of the EC-Gre-Stl complex that explains the observed Stl activities and provides insight into possible cooperative action of secondary channel factors and other antibiotics binding at the Stl pocket. These results offer a new strategy for high-throughput screening for prospective antibacterial agents.
AB - Antibiotic streptolydigin (Stl) inhibits bacterial transcription by blocking the trigger loop folding in the active center of RNA polymerase (RNAP), which is essential for catalysis. We use acoustic force spectroscopy to characterize the dynamics of transcription elongation in ternary elongation complexes (ECs) of RNAP in the presence of Stl at a single-molecule level. We found that Stl induces long-lived stochastic pauses while the instantaneous velocity of transcription between the pauses is unaffected. Stl enhances the short-lived pauses associated with an off-pathway early paused state intermediates of the RNAP nucleotide addition cycle. Unexpectedly, we found that transcript cleavage factors GreA and GreB, which were thought to be Stl competitors, do not alleviate the Stl-induced pausing; instead, they synergistically increase transcription inhibition by Stl. This is the first known instance of a transcriptional factor enhancing antibiotic activity. We propose a structural model of the EC-Gre-Stl complex that explains the observed Stl activities and provides insight into possible cooperative action of secondary channel factors and other antibiotics binding at the Stl pocket. These results offer a new strategy for high-throughput screening for prospective antibacterial agents.
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U2 - 10.1093/nar/gkae1135
DO - 10.1093/nar/gkae1135
M3 - Article
C2 - 39656915
SN - 0305-1048
VL - 53
JO - Nucleic acids research
JF - Nucleic acids research
IS - 1
M1 - gkae1135
ER -