Student Theses and Dissertations


James Chen

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Darst Laboratory


In bacteria, a single RNA polymerase (RNAP) performs all transcription. The overall structure of bacterial RNAP resembles a crab claw with pincers comprising the β’ and β subunits and a large cleft where the active site sits. Structural information about this essential enzyme has mainly been provided by X-ray crystal structures of stable transcription complexes. RNAP crystal structures are difficult to obtain and the captured states may not always represent physiological states of the enzyme due to crystal packing artifacts. Recent advances in electron detectors and software enable near-atomic resolution structures of large biological complexes to be determined by single particle cryo-electron microscopy (cryo-EM). Unlike X-ray crystallography, cryo-EM samples can be directly visualized without crystallization. In this thesis, I optimized and utilized cryo-EM methodologies to structurally characterize several bacterial RNAP complexes from Escherichia coli (Eco) and Mycobacterium tuberculosis (Mtb): (1) Eco σ70-holoenzyme (Eσ70) in complex with the Eco non-coding RNA (ncRNA) 6S RNA; (2) Mtb RNAP bound to the RNAP inhibitor Fidaxomicin (Fdx); (3) Eσ70 bound to the F element-encoded TraR protein; and (4) Eσ70-dependent promoter DNA melting intermediates stabilized by the TraR transcription factor. Using cryo-EM, I captured RNAP structures that were intractable to crystallization, visualized multiple RNAP conformational states populated in solution, deconvoluted RNAP molecular motions, and observed transient complexes. The work in this thesis showcases the power of cryo-EM to examine macromolecular machines in action. (1) Bacterial 6S RNAs globally regulate transcription by RNAP, directly competing with promoter DNA binding. During transitions between exponential and stationary growth phases, Eco 6S RNA plays a key role in the transcriptional reprogramming by interacting specifically with the housekeeping Eσ70. During my initial cryo-EM experiments with the Eco 6S RNA-Eσ70 complex, I encountered a severe particle orientation bias in my samples. I discovered that the zwitterionic detergent 3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO) was uniquely effective at solving this issue for bacterial RNAPs. Using this detergent, I determined the cryo-EM structure of the Eco 6S RNA/Eσ70 complex in combination with footprinting and crosslinking approaches in order to elucidate the structural mechanism of 6S RNA mediated inhibition of Eσ70. The structure reveals that 6S RNA is composed of duplex RNA segments that have A-form C3'-endo sugar puckers but with widened major groove widths, giving the RNA an overall architecture that mimics B-form promoter DNA. The results showed how 6S RNA specifically targets Eσ70 and how an ncRNA can mimic B-form DNA to directly regulate transcription by the DNA-dependent RNAP. (2) Fdx is a RNAP-binding drug that is highly effective against Mtb RNAP in vitro. In collaboration with postdoctoral fellow Hande Boyaci, we solved cryo-EM structures of Mtb σA-holoenzyme (σA-holo) bound to the actinobacteria general transcription factor RbpA and an upstream promoter DNA fork (us-fork) in the presence and absence of Fdx to identify the structural determinants of Fdx binding to RNAP. The results show that Fdx acts like a doorstop fo jam the RNAP clamp module in an open conformation. The structures define the Fdx binding pocket, which includes contacts with RbpA, explaining the drug's strong effect on Mtb. (3) Under starvation conditions, Eco changes the expression of almost one-quarter of its genes within 5 minutes. This global response depends on two factors, the alarmone ppGpp, and the transcription factor DksA. Unlike most transcription factors, ppGpp and DksA bind directly to RNAP but not to DNA. Another transcription factor, TraR, regulates RNAP the same way as ppGpp and DksA. TraR, and ppGpp/DksA, can activate or inhibit transcription initiation depending on the promoter sequence. TraR inhibits Eσ70-dependent transcription from ribosomal RNA and ribosomal protein promoters and activates amino acid biosynthesis and transport promoters in vivo and in vitro. To understand how these factors modulate transcription initiation, I solved cryo-EM structures of Eσ70, with and without TraR, and an open promoter complex (RPo) using a ribosomal promoter that is regulated by TraR, rpsT P2. My cryo-EM structural analyses show that TraR regulates Eσ70 transcription initiation by binding and inducing major conformational changes to mobile RNAP domains that affect RNAP-promoter DNA interactions. (4) Transcription initiation is a multi-step process that leads to the formation of RPo. Using TraR to modulate Eσ70 transcription initiation on the ribosomal protein promoter rpsT P2, I observed promoter DNA melting intermediates by cryo-EM. These structures and supporting biochemical data delineate steps of the RPo formation pathway and provide insight into transient promoter-RNAP interactions that occur along this pathway. These structures span the initial recognition of the duplex promoter in a closed complex (RPc) to the final RPo. Findings from this thesis provide structural and biochemical insight into bacterial RNAP regulation and function. The work in this thesis shows how single-particle cryo-EM can be applied to study transcription factors and small molecules that modulate RNAP as well as transient transcriptional states. This thesis research outlines a framework for future studies of bacterial transcription complexes using cryo-EM.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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