1. Regulation of transcription by protein TFs and coupling of transcription and translation.
Project 1: Structure and function of paused RNAP bound to NusA.
NusA is an essential bacterial TF. It increases RNAP pausing, facilitates RNA folding, and stimulates termination. Despite 40 years of work mechanistic details were lacking. We purified and biochemically characterized a paused RNAP with a nascent RNA transcript containing a pause stabilizing hairpin and NusA. High-resolution reconstructions allowed us to propose a role for NusA, observe a folded RNA structure in the context of RNAP, and see how catalysis is inhibited in the paused state (Guo et al., Mol. Cell 2018 - results featured on the cover. Fig. 1A).
Project 2: Structural basis of RNAP backtracking and reactivation.
RNAP backtracks along the DNA after misincorporations or to prolong a pause. Backtracking requires reactivation. TFs like GreA or GreB stimulate RNA cleavage so transcription can resume and to remove erroneous bases after misincorporations. We obtained four high-resolution structures representing: i) a backtracked complex; ii) a GreB bound backtracked complex before RNA cleavage; iii) a GreB bound complex after RNA cleavage; and iv) a reactivated, substrate bound complex. The structures provided new insights into the reaction mechanism, the dynamic nature of the process and we proposed a comprehensive model for the role of GreB (Abdelkareem et al., Mol. Cell 2019, Fig. 1B). Similar work has been done for GreA and we are currently preparing a manuscript.
Project 3: Structural basis of transcription translation coupling
Transcription and translation are functionally coupled in bacteria. The ribosome initiates translation on the nascent RNA and it had been proposed that it is physically coupled to RNAP almost 60 years ago. However, the structural basis was unclear. We obtained high-resolution reconstructions of a supramolecular complex between RNAP and the ribosome (expressome) in several functional states: an uncoupled expressome, an expressome coupled by the TF NusG, and a collided expressome. This was a breakthrough for my team and resolved previous controversies (Webster et al., Science 2020, Fig. 1C).
2. Role of regulatory RNAs in transcription.
Project 4: Structural basis of transcription regulation by RNA
Non-coding RNAs also regulate RNAP but this is a poorly understood phenomenon. We studied an RNA called putL, which renders RNAP resistant to pause and termination signals. We biochemically studied RNAP complexes regulated by putL and purified them for cryo-EM. The reconstructions allowed us to see how putL changes RNAP to become less responsive to pause signals. We also used putL as a tool to structurally characterize intermediates in transcription termination, which is still incompletely understood. The results will clarify open questions and we are currently preparing a manuscript.
3. Role of RNAP conformational changes.
Project 5:
NusG is a TF, which suppresses pausing. However, it binds RNAP along with NusA, which increases pausing. Structures of RNAP ECs bound to NusA, or NusG, or both show how NusG shifts the RNAP conformational equilibrium to an active state, while NusA shifts it to a paused state. When both TFs are bound, RNAP adopts an intermediate state. Thus, TFs modulate RNAP conformation and consequently the response to pause signals. We have also further characterized the roles of NusA and NusG during transcription termination and our results are currently under revision.
To summarize, we used high-resolution cryo-EM and biochemistry to study complexes with fundamental roles in gene expression. We were able to answer a number of open questions in the transcription field and raise many news ones.
