Periodic Reporting for period 4 - TRANSREG (Structural and biochemical studies on the regulation of transcription during elongation)
Periodo di rendicontazione: 2020-12-01 al 2021-05-31
DNA stores genetic information, which is the blueprint for an organism. It is retrieved in two steps in a process called gene expression. First, DNA is transcribed into RNA by RNA polymerase (RNAP). Second, the RNA is translated by the ribosome to synthesize proteins. Gene expression and its regulation are incompletely understood and the key enzymes are universally conserved. Thus, studies in one species are relevant for our understanding of gene expression in all domains of life.
Transcription is tightly regulated because it affects many aspects of biology and misregulation causes severe human diseases (e.g. cancer, autoimmunity, or neurological disorders). In addition, bacterial RNAP is a prime drug target against infectious diseases. A detailed understanding of transcription is crucial because of its fundamental biological role, to develop strategies against abnormal regulation, and to develop new antibiotics.
Structural biology is used to study enzymes at atomic resolution and obtain mechanistic insights of biological processes, which is needed for rational drug design. High-resolution structures of functional RNAP complexes revolutionized our understanding of transcription and paved the way to address more complex questions regarding its regulation. Transcription is frequently interrupted by pauses, which are modulated by transcription factors (TFs) and regulate gene expression. We addressed the following questions in this project:
i) How do protein TFs modulate pausing?
ii) How are RNAP and the ribosome coupled in bacteria?
iii) How do regulatory RNAs modulate pausing?
iv) How are TFs influencing RNAP conformation during pausing or elongation?
We combined biochemistry and electron cryo-microscopy (cryo-EM) to take snapshots of active RNAP during regulatory events and better understand the entire process.
The ERC Starting Grant allowed the team to focus on the questions outlined above and publish our results (Guo et al., Mol. Cell 2018; Abdelkareem et al., Mol. Cell 2019; Webster et al., Science 2020). Furthermore, we are finalizing a number of additional projects, which benefitted from ERC funding.
Transcription is tightly regulated because it affects many aspects of biology and misregulation causes severe human diseases (e.g. cancer, autoimmunity, or neurological disorders). In addition, bacterial RNAP is a prime drug target against infectious diseases. A detailed understanding of transcription is crucial because of its fundamental biological role, to develop strategies against abnormal regulation, and to develop new antibiotics.
Structural biology is used to study enzymes at atomic resolution and obtain mechanistic insights of biological processes, which is needed for rational drug design. High-resolution structures of functional RNAP complexes revolutionized our understanding of transcription and paved the way to address more complex questions regarding its regulation. Transcription is frequently interrupted by pauses, which are modulated by transcription factors (TFs) and regulate gene expression. We addressed the following questions in this project:
i) How do protein TFs modulate pausing?
ii) How are RNAP and the ribosome coupled in bacteria?
iii) How do regulatory RNAs modulate pausing?
iv) How are TFs influencing RNAP conformation during pausing or elongation?
We combined biochemistry and electron cryo-microscopy (cryo-EM) to take snapshots of active RNAP during regulatory events and better understand the entire process.
The ERC Starting Grant allowed the team to focus on the questions outlined above and publish our results (Guo et al., Mol. Cell 2018; Abdelkareem et al., Mol. Cell 2019; Webster et al., Science 2020). Furthermore, we are finalizing a number of additional projects, which benefitted from ERC funding.
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.
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.
NusA had been studied since the 1970s but we lacked a structural understanding of its role during pausing. Our results explain how catalysis is halted, and how NusA stimulates pausing and RNA folding. Furthermore, by studying NusA and NusG in the context of an active EC, we were able to see competitive and cooperative effects of TFs during transcription elongation and termination.
Our work on GreB (and GreA) provides snapshots and follow RNAP along the reactivation pathway after backtracking. Using cryo-EM, we were able to study fully functional complexes and provide insights with implications for transcriptional fidelity.
Coupling of RNAP to the ribosome had been suggested by Marshall Nirenberg in 1964. The TF NusG had been proposed to bridge RNAP and the ribosome but recent data contradicted those results. Our structures of RNAP and ribosome on a shared transcript resolved earlier controversies and showed the entire machinery, which executes gene expression in bacteria, at near-atomic resolution. The structures suggest functional roles for physical coupling and also raised many new questions. Along with results from other labs, it sparked renewed interest in the subject of transcription translation coupling.
The structural basis for RNAP regulation by RNA received less attention. Several RNAs are known to regulate RNAP directly. We obtained high-resolution structures of a regulatory RNA bound to RNAP and understand how an RNA stimulates its own synthesis. The structures also provided insights on transcription termination, a poorly understood process.
Our work on GreB (and GreA) provides snapshots and follow RNAP along the reactivation pathway after backtracking. Using cryo-EM, we were able to study fully functional complexes and provide insights with implications for transcriptional fidelity.
Coupling of RNAP to the ribosome had been suggested by Marshall Nirenberg in 1964. The TF NusG had been proposed to bridge RNAP and the ribosome but recent data contradicted those results. Our structures of RNAP and ribosome on a shared transcript resolved earlier controversies and showed the entire machinery, which executes gene expression in bacteria, at near-atomic resolution. The structures suggest functional roles for physical coupling and also raised many new questions. Along with results from other labs, it sparked renewed interest in the subject of transcription translation coupling.
The structural basis for RNAP regulation by RNA received less attention. Several RNAs are known to regulate RNAP directly. We obtained high-resolution structures of a regulatory RNA bound to RNAP and understand how an RNA stimulates its own synthesis. The structures also provided insights on transcription termination, a poorly understood process.