Periodic Reporting for period 1 - TF3C_EM (Structure-function studies of the general transcription factor IIIC (TFIIIC))
Reporting period: 2017-03-01 to 2019-02-28
In eukaryotes, the task of transcription is shared by three types of RNA polymerases, selected depending upon the gene being read. tRNA gene (tDNA) transcription begins with the recruitment of the multi-protein transcription factor IIIC (TFIIIC) to two characteristic gene-internal promoter elements named A- and B-boxes. This sets the stage for the assembly of the pre-initiation complex which also includes transcription factor IIIB and RNA polymerase III and thereby, the start of transcription. However, the lack of information on the overall TFIIIC structure and its DNA binding mechanism has hampered the understanding of transcription initiation. ‘TF3C_EM’ focused on the structure-function elucidation of TFIIIC with the following objectives:
(1) Characterization of the molecular architecture of TFIIIC,
(2) Understanding the tDNA recognition mechanism by TFIIIC and
(3) Understanding the dynamics between the two subdomains of TFIIIC- τA and τB.
I obtained a low resolution structure of TFIIIC, purified from yeast using the TAP-tag strategy. I was also able to study the complete pre-initiation complex, paving the way for further understanding the transcription initiation. Moreover, ‘TF3C_EM’ gave me an opportunity to widen my skill set from working with different model organisms (yeast, insect cells), to handling high-end cryo-electron microscopes, data collection and processing. I was also able to build on my project management and science communication skills at EMBL during this time. These skills are extremely valuable for my future position.
Alongside I studied the complete pre-initiation complex (PIC) comprising of TFIIIC, Brf1-TBP component of TFIIIB, RNA Polymerase III (Pol III) and DNA. With the minimal PIC now characterized (Vorländer et al, 2018), studying TFIIIC interactions with Pol III has been made feasible.
In total, I have studied the TFIIIC holocomplex for cryo-EM studies and with some preliminary experiments I have also explored the TFIIIC holocomplex structure in the bigger context of a transcription initiation complex. With ‘TF3C_EM’ I reinforced my ability to develop my own projects, work independently with a good balance in leadership and team-spirit.
During the time of the ‘TF3C_EM’ fellowship, I disseminated my results amongst the peers at international conferences - ‘Conference on methods and applications in the frontier between MX and CryoEM’ in 2017 in Barcelona, Spain being one of them. Here I discussed the technical aspects of my project with pioneers in the field of cryo-EM and modelling in X-ray crystallography. I also qualified for attending ‘The Lindau Noble Laureate meeting 2018’ (LiNo18), Lindau, Germany which was an ecstatic mix of people from various fields in biology. This ‘one-week’ long meeting was an amazing platform to discuss research with Nobel laureates and young scientists. The attendees came from various fields including physics, chemistry, biology and physiology. I was nominated as a Marie-Curie fellow and also had the opportunity to network with other fellows at the LiNo18.
I also attended the ‘Workshop on advanced topics in EM structure determination: challenges and opportunities’ organized by NRAMM in 2017 at the New York Structural Biology Center. There, I was able to build upon my cryo-EM expertise and network with some of the bright minds in the field. ‘TF3C_EM’ has given me the opportunity to not only build on my scientific expertise but also on my science communication skills with the above mentioned conferences and on-site courses at EMBL. This entire skill set is highly relevant for my future endeavors as an independent researcher.
Moreover, since the start of TF3C_EM project, I have been involved in disseminating results of the project to my peers within EMBL and at international conferences. Alongside, I have also been active in outreach to the society along with the European Learning Laboratory for the Life Sciences (ELLS) program at EMBL. ELLS is an initiative where high school students and teachers get to interact with scientists. As an EMBL school ambassador, I presented my work at AKG Bensheim to the local audiences. It was an enthralling experience to meet and discuss scientific research with high school students (http://emblog.embl.de/ells/embl-school-ambassadors/embl-school-ambassador-diaries/heena-khatters-school-ambassador-diary/). Also, I have been involved with teacher training programs (learningLABs) at the EMBL, where high-school teachers come from all over Europe and get hands-on training in basic molecular biology.
I also actively participated in the science fair “Explore Science” at Mannheim where the EMBL team presented basic science in a fun way. We had ‘cell cookies’, microscopes to show the bacterial growth, a puzzle to differentiate eukaryotic and prokaryotic cell amongst other things for the general public. It was an amazing experience to discuss basic research and also answer general concerns. Thus, in total as a Marie-Curie fellow I had the opportunity to discuss my research with public and also promote basic research amongst students. Alongside, I could also build on my soft skills of project management and science communication at EMBL with the courses offered onsite. These skills coupled with my acquired scientific expertise are extremely valuable for my future position as a researcher in an industrial set-up where I will apply cryo-EM for drug development.