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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - TRANSREG (Structural and biochemical studies on the regulation of transcription during elongation)

Teaser

Summary

Every organism stores genetic information in the form of DNA. This information fulfils two major roles: i) It serves as the organismâ€™s blueprint; and ii) It allows an organism to respond and adapt to changes in the environment. Thus, to develop on the one hand and respond to the environment on the other hand, every organism needs to retrieve the information stored in its DNA. This fundamental process is called gene expression and proceeds in two steps: The first one, where DNA is copied into RNA, is called transcription and is carried out by a universally conserved molecular machine called RNA polymerase (RNAP). In the second step, the RNA copy is used as a template to synthesize proteins in a process termed translation. Transcription is a fundamental, yet incompletely understood process. The key player, RNAP, is universally conserved. This means studies on this machinery in one species are relevant for the general understanding of gene expression in all domains of life.
Due to the fundamental role, gene expression, and in particular transcription, needs to be tightly regulated and coordinated. Accordingly, transcription and its regulation affect essentially every aspect of biology. Misregulation on the other hand causes numerous human diseases including cancer, autoimmunity, neurological disorders, diabetes, cardiovascular diseases and obesity. To develop successful strategies against abnormal regulation, it is imperative to unravel the processes at work in detail.
In addition, a detailed understanding of the bacterial transcription machinery in particular, has important biomedical implications because RNAP is one of the prime drug targets to counter infectious diseases. Resistance to antibiotics is rising all across the globe and poses a serious medical concern. A mechanistic understanding of the transcription process is thus crucial not only because of its fundamental biological importance but also because of the biomedical implications.
Structural biology provides tools to take snapshots of important molecular machines and visualize them at atomic resolution. We require this level of understanding to obtain mechanistic insights of a biological process and comprehend it at the molecular and chemical level. The same detailed understanding is necessary for structure-based drug design.
High resolution structures of RNAP core as well as RNAP elongation complexes have revolutionized our understanding of transcription and allowed us to interpret transcription at the molecular level. This groundwork paved the way to address more complex questions regarding the regulation of transcription. Transcriptional pausing, a temporary interruption of transcription, plays a major role in regulating gene expression in pro- and eukaryotes. A full and mechanistic understanding is crucial and requires a combination of structural and biochemical studies. This is where our team is contributing.
The general theme of research in our team is the regulation of transcription by protein and non-coding RNA factors. Currently we focus on three major questions:
1. How do protein transcription factors modulate the rate of transcription?
2. How do non-coding RNA molecules modulate transcription rates?
3. What is the role of RNA polymerase dynamics and conformational changes during transcription?

To obtain answers to these questions we combine biochemistry and high-resolution structural biology. The main tool is single particle electron cryo-microscopy (cryo-EM), which allows us to take snapshots of active RNAP during regulatory events. We combine these high-resolution structures with biochemical results to gain a mechanistic understanding of the entire process.
Through financial support from the ERC Starting Grant, the team started work on all three questions outlined above. This provided us with the freedom to completely focus on our goals and gave rise to our first published results earlier last year (Guo et al., Mol. Cell 2018). In addition, we made

Work performed

The initial period of the project was used to setup purification and biochemical assays to characterize functional complexes of RNAP in complex with protein and RNA transcription factors. At the same time, we transitioned from using X-ray crystallography as our primary method to obtain high-resolution structural information to single particle cryo-EM. This was the right decision given the recent advances in cryo-EM (which is also reflected in the 2017 Nobel Prize in Chemistry). Obtaining high resolution structures has now become easier and less time-consuming.

Aim 1 - IN PROGRESS: Regulation of transcription elongation by protein factors.
We proposed to use a combination of X-ray crystallography, single particle cryo-EM, and biochemistry to study the role of general transcription factors, which modulate transcription rates. Specifically, we proposed to study RNA polymerase (RNAP) elongation complexes (EC) bound to transcription factors such as bacterial NusA, which are involved to regulate RNAP directly.

Project 1: Structure and function of paused RNAP bound to NusA.
NusA is an essential bacterial transcription factor with a wide range of roles. It stabilizes RNAP in a paused state, facilitates RNA folding, stimulates termination and paradoxically can also prevent termination in complex with additional factors. Because of its wide range of roles, it has been studied for over 40 years but mechanistic details were lacking. Originally, we proposed to combine X-ray crystallography and single particle cryo-EM but we decided to focus on EM exclusively. After biochemical characterization, we purified a functional E. coli RNAP EC assembled on a pause-inducing DNA sequence. The complex contained an RNA transcript, which forms a pause stabilizing hairpin structure, and NusA. We obtained high-resolution reconstructions using a Titan KRIOS microscope, which allowed us to propose a role for NusA in stabilizing the paused state. We also observed for the first time what appears to be a novel translocation intermediate, which explains why catalysis is inhibited in the paused state. Finally, we observed for the first time a nascent, folded RNA hairpin structure accommodated in the RNAP exit channel. The results of this work have been summarized in an article, which was published in Molecular Cell and featured on the cover (Guo et al., Mol. Cell 2018).

Project 2: Structural basis of RNAP backtracking and reactivation.
We are also interested in a transcription factor called GreB. GreB is involved in rescuing backtracked and arrested RNAP. RNAP backtracks along the DNA template and extrudes the RNA 3â€™-end from the active site as a result of misincorporations or to stabilize a paused state. Backtracking may cause long-lived pauses or even complete arrest of the enzyme, which requires reactivation. Transcription factors like GreB can cleave the RNA 3â€™-end to produce a shortened RNA, which is newly aligned with the active site so transcription can resume. In the case of misincorporations, this also removes the erroneous base from the transcript. Using single particle cryo-EM we obtained four high-resolution reconstructions that cover the entire reaction: 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 provide new insights into the reaction mechanism, the dynamic nature of the process and allow us to propose a comprehensive model for the role of GreB.

We also made progress concerning other functional RNAP elongation complexes bound to conserved transcription factors, which modulate transcription rates. We have a number of high-resolution reconstructions that are currently built and interpreted.

Aim 2 - IN PROGRESS: Role of regulatory RNAs in transcription.
Analogous to protein transcription factors, we proposed to address the role that non-coding RNAs play in regulating transcription. So far, we f

Final results

The transition from X-ray crystallography to single particle cryo-EM is revolutionizing structural biology. For the first time we are able to observe macromolecules with key roles in biology close to their native state. It also allows us to obtain structures of important intermediates more easily and, using computational tools, estimate conformational heterogeneity in an ensemble of molecules. In the current projects, these advantages were key to further our understanding of regulatory processes during transcription.
Regulation of transcription elongation by protein factors.
The role of the bacterial transcription factor NusA in regulating RNA polymerase (RNAP) has been studied since the early 1970s. In our cryo-EM reconstruction of a paused E. coli RNAP NusA complex we observe for the first time: (1) How the factor binds to RNAP as well as a novel interaction, which was biochemically verified; (2) How NusA provides a positively charged environment that likely facilitates folding of RNA structures; (3) A novel RNAP conformation, which explains how transcription is temporarily inhibited; (4) How NusA can rotate relative to RNAP, a feature likely crucial to fulfil its diverse roles; (5) A folded RNA structure within a channel of RNAP. The latter is important because transcription termination, which is poorly understood on a mechanistic level, can also be triggered by similar structures and is stimulated by NusA.
When RNAP transcribes DNA into RNA it moves unidirectional relative to the DNA template. However, RNAP may halt as a result of an error or due to regulatory DNA sequences. Both can trigger backtracking, where RNAP moves backward relative to the DNA and blocks its catalytic site. Cleavage of a short piece of RNA from the end of the transcript reactivates RNAP and transcription resumes. Cleavage is accelerated by transcription factors such as bacterial GreB. Backtracking occurs in all species and serves at least two roles: (1) It allows error correction (proof-reading) and increases transcriptional fidelity, a very important phenomenon; (2) It stabilizes paused states of RNAP, which are used to regulate transcription rates or recruit transcription factors. Using single particle cryo-EM, we obtained high-resolution snapshots of (i) backtracked RNAP, where we can observe how the enzyme is blocked for catalysis; (ii) backtracked RNAP in complex with GreB before RNA cleavage, where we can see for the first time how the RNA substrate is involved to stabilize an active site ready for cleavage; (iii) RNAP bound to GreB after RNA cleavage; and (iv) reactivated RNAP with a bound substrate in the active site, which shows why GreB needs to leave the active site so transcription can resume.
We are continuing our efforts to obtain insights into the roles of protein transcription factors and have high-resolution reconstructions of functional RNAP in complex with multiple factors bound simultaneously. In the past, many studies focused on RNAP in isolation or RNAP bound to one transcription factor. However, quantitative studies on the E. coli proteome as well as genome wide studies on transcription factor distributions indicate that most if not all RNAP molecules are likely bound to several transcription factors at the same time (Mooney et al., JMB 2009, Schmidt et al., Nat. Biotechnol. 2016). Thus, studying the interplay of synergistic or antagonistic transcription factors bound to RNAP is a more accurate reflection of the situation in vivo.
Regulation of transcription elongation by RNA.
A phenomenon, which has received relatively little attention is the regulation of RNAP by RNA. Several RNA elements have been described in the literature to regulate RNAP directly. A prominent example is bacterial 6S RNA and recent publications provided insights into its role (Chen et al., Mol. Cell 2017). Next to nothing is known about RNA molecules that affect RNAP during transcription elongation. This is where our team will contribute. After biochemic