For decades, scientists have known that two unusual classes of antibiotics can shut down RNA polymerase, the molecular machine responsible for copying genetic information from DNA into RNA. The compounds, known as CBR9379 and AAP-SO2, act against RNA polymerases from Escherichia coli and Mycobacterium tuberculosis, respectively. Yet researchers could not fully explain how these drugs stopped the enzyme. They knew where the antibiotics attached, but not what essential process they disrupted. A new study from Rockefeller University, published in the Proceedings of the National Academy of Sciences, has now revealed the answer: the antibiotics freeze a moving component of RNA polymerase, preventing it from supporting the chemical reaction required to build RNA.
The discovery resolves a long-standing mystery in antibiotic biology while exposing an unexpected feature of one of the most fundamental enzymes in life. RNA polymerase is essential because it initiates transcription, the first step in gene expression. During transcription, the enzyme travels along a DNA template and links together RNA nucleotides in a precise sequence. Every new nucleotide must be selected, positioned and chemically joined to the growing RNA chain. The enzyme is not a rigid molecular scaffold during this process. Instead, it repeatedly changes shape, cycling through a series of conformations that allow it to capture substrates, catalyze bond formation and then move forward along the DNA.
One of the best-known moving elements in bacterial RNA polymerase is the trigger loop, a flexible structure that closes over the active site during nucleotide addition and reopens afterward. When the correct RNA building block enters the active site, the trigger loop folds into a closed configuration that helps align the nucleotide and stabilize the reaction. It then withdraws to permit the enzyme to advance to the next position. This repeated opening and closing is central to the nucleotide addition cycle. Until now, however, scientists had not understood the precise role of another nearby structure, called the rim helices or F-loop. Its location suggested that it might influence the active site, but whether it moved during transcription—and why—remained uncertain.
The research team, led by Seth A. Darst at Rockefeller University, used cryo-electron microscopy to observe RNA polymerase in multiple functional states. Unlike conventional X-ray crystallography, which generally provides a highly detailed but relatively static image of a protein, cryo-electron microscopy can capture large populations of molecules adopting different shapes. By collecting thousands of images and sorting them according to structural features, researchers reconstructed a molecular ensemble showing how RNA polymerase shifts between conformations while synthesizing RNA. The experiments examined enzymes from both E. coli and M. tuberculosis, allowing the investigators to determine whether the suspected movement was limited to one bacterial species or represented a more broadly conserved mechanism.
The images confirmed the expected behavior of the trigger loop, which alternated between open and closed positions. They also revealed that the neighboring rim helices/F-loop undergoes a coordinated motion. As the trigger loop closes over the active site, the rim helices/F-loop swings inward and briefly contacts it. This interaction appears to stabilize the closed catalytic configuration, helping RNA polymerase maintain the precise geometry needed to add the next nucleotide. Once the chemical step is completed, the structures can separate and the enzyme can reset. The finding means that RNA polymerase relies on a previously unrecognized partnership between two mobile elements rather than on the trigger loop acting alone.
The antibiotics exposed this hidden mechanism by disrupting it. In untreated samples, RNA polymerase molecules occupied both open and closed conformations, reflecting the natural transitions that occur during transcription. When CBR9379 or AAP-SO2 was added, the closed conformation disappeared. Both drugs locked the rim helices/F-loop in an open position, even though they bind at chemically distinct sites in the bacterial enzyme. Unable to swing inward and contact the trigger loop, the F-loop could no longer stabilize the active site. The trigger loop was therefore prevented from carrying out the productive motion required for nucleotide addition, and RNA synthesis came to a halt.
This shared mechanism is notable because the two antibiotics are chemically unrelated and act on RNA polymerases from different bacterial species. CBR9379 targets the enzyme in E. coli, a widely used model organism, while AAP-SO2 targets the corresponding enzyme in M. tuberculosis, the pathogen that causes tuberculosis. Their ability to produce the same structural outcome suggests that the moving rim helices/F-loop is not an incidental feature of one bacterial enzyme. Instead, it may represent a fundamental requirement for efficient transcription across diverse organisms. The researchers also suggest that related motions could occur in other cellular RNA polymerases, although the details may vary among bacteria, archaea and eukaryotes.
The work illustrates how antibiotics can function as probes of basic biology. Scientists initially regarded these compounds mainly as inhibitors whose therapeutic potential depended on their ability to stop bacterial growth. By showing exactly which conformational transition the drugs suppress, the study clarifies how bacterial transcription can be interrupted at a structural level. The findings also demonstrate why understanding protein motion is essential in drug research. A binding site alone does not always reveal an inhibitor’s mechanism. In this case, the decisive event was not simply that the antibiotic occupied a pocket, but that it prevented a flexible element from moving into the position needed for catalysis.
The results could guide the design of future antibiotics, particularly against tuberculosis. Drug-resistant M. tuberculosis strains are increasingly difficult to treat, and resistance to rifampicin, a cornerstone of tuberculosis therapy, remains a major clinical challenge. AAP-SO2-like compounds inhibit RNA polymerase through a mechanism distinct from rifampicin, creating the possibility of combining drugs that attack the same essential enzyme in different ways. Earlier work has suggested that such compounds may act alongside rifampicin in more effective treatment combinations. The newly identified F-loop motion provides medicinal chemists with a structural target: compounds could be optimized to stabilize the inactive open state or otherwise prevent the loop from engaging the trigger loop.
The study ultimately turns a molecular snapshot into a dynamic explanation of transcription. RNA polymerase is now seen not merely as a machine with a fixed catalytic pocket, but as an ensemble of shifting structures whose coordinated movements determine whether RNA synthesis proceeds. By trapping the enzyme in one of those states, CBR9379 and AAP-SO2 reveal a vulnerability that bacteria may find difficult to bypass without compromising a process essential for survival. The discovery of this active-site motion deepens scientists’ understanding of gene expression and offers a blueprint for developing antibiotics capable of disabling pathogens through a new and highly specific mechanism.
Subject of Research: RNA polymerase dynamics, transcription, and antibiotic inhibition
Article Title: RNA polymerase inhibitors reveal active-site motions essential for the nucleotide addition cycle
Web References: https://www.pnas.org/doi/10.1073/pnas.2609228123 ; https://www.rockefeller.edu/our-scientists/heads-of-laboratories/959-seth-a-darst/ ; https://www.darstcampbell.com/
References: Proceedings of the National Academy of Sciences; DOI: 10.1073/pnas.2609228123
Image Credits: Laboratory of Molecular Biophysics at The Rockefeller University
Keywords: RNA polymerase, transcription, antibiotics, CBR9379, AAP-SO2, tuberculosis, E. coli, Mycobacterium tuberculosis, cryo-electron microscopy, trigger loop, rim helices, F-loop, drug resistance
Tags: antibiotic action on bacterial RNA polymeraseantibiotics targeting RNA synthesisDNA to RNA transcription processenzyme conformational dynamics in RNA polymeraselong-standing mystery in antibiotic biologymolecular mechanism of CBR9379 and AAP-SO2molecular structure of RNA polymerase inhibitorsrecent discoveries in transcription biologyregulation of transcription initiationRNA polymerase inhibition mechanismRNA synthesis and gene expressionRockefeller University RNA research
