Publications

News about the Mechanisms and Evolution of Transcription!

Transcription is carried out by evolutionary conserved RNA polymerases and subject to regulation by different strategies. The control of individual genes is enabled by a plethora of DNA-binding transcription factors that respond to changes in the environment and enable the up- or downregulation gene expression; the structural basis and mechanisms of genespecific regulation has been characterised in great detail. Much less is known about the global regulation of transcription that is enabled by RNAP-binding factors. Two research articles from the ISMB RNAP laboratory published back-to-back in Nature Communications explore and characterise the global control of the archaeal RNAP in two different scenarios. Firstly, a multidisciplinary study combining functional genomics with in vitro transcription assays hints at a paradigm shift for the regulation of transcription in Archaea (Blombach et al., 2021), and secondly, a cryo-EM analysis elucidates, for the first time, the structural basis of RNAP inhibition by repressors involved in the archaeal virus-host arms race (Pilotto et al., 2021).
Dr Fabian Blombach, the lead author of the first study explains: ‘The current paradigm in the field dictates that transcription regulators positively or negatively interfere with the recruitment of RNA polymerase to the promoter at the stage of initiation. We challenged this hypothesis using a functional genomics approach by mapping the dynamic (re-)distribution of RNAP correlating it with the cellular RNA levels, genome-wide and at single nucleotide resolution. We could show that it is not only simple ‘access’ of RNAP to the promoter that determines the expression level of a gene, but sophisticated mechanisms that occur during early elongation (Fig. 1). The factors Spt4/5 and Elf1 are successively recruited to the transcription elongation complex before the RNA polymerase makes a transition into productive transcription. Quite surprisingly, we also found that RNA polymerases in the early elongation phase recruit a ribonuclease, the transcription termination factor aCPSF1, that negatively regulates transcription likely by RNA cleavage-induced premature termination mechanism, a mode of regulation that is evolutionary conserved in bacteria and eukaryotes.’

Dr Simona Pilotto, the lead author of the second study elaborates on her work: The inhibition of RNAP and the resulting attenuation of the transcriptome plays a crucial role in the interaction between viruses and their hosts during infection. My aim is to understand the structural basis of ‘switching off’ RNAP, as this is highly relevant for the design of novel antibiotics and antiviral drugs. I have solved the cryo-EM structures of the complexes formed between the Sulfolobus acidocaldarius RNAP and two distinct regulators (RIP and TFS4), and my structures provide insights into the detailed mechanisms underlying the very potent inhibition (Fig. 2). RIP is encoded by the Acidianus two-tailed virus (ATV) and sterically interferes with the interaction of template DNA and transcription factors by molecular mimicry of initiation factors. TFS4 is encoded by the Sulfolobus host and is expressed in response to infection with the Sulfolobus Turreted Icosahedral Virus (STIV). TFS4 is related to elongation factors including TFIIS and targets the RNAP NTP entry pore through which it inactivates RNAP in an allosteric fashion by inducing a widening of the DNA-binding channel disrupting the bridge helix and trigger loop active site motifs. The most intriguing conclusion of my work is that the inhibitory strategies and mechanisms reveal the underlying functional conservation
of RNAPs: unrelated inhibitors have evolved to exploit factor- and nucleic acid binding sites, and conformational flexibilities that are intrinsic to all RNAPs to effectively repress its activity.

Blombach, F., Fouqueau, T., Matelska, D., Smollett, K., and Werner, F. (2021). Promoter-proximal elongation regulates transcription in archaea. Nat Commun 12, 5524.
Pilotto, S., Fouqueau, T., Lukoyanova, N., Sheppard, C., Lucas-Staat, S., Diaz-Santin, L.M., Matelska, D., Prangishvili, D., Cheung, A.C.M., and Werner, F. (2021). Structural basis of RNA polymerase inhibition by viral and host factors. Nat Commun 12, 5523.

Posted by ubcg49z in Lab news, News, Publications

Professor Steven Perkins’ paper featured on the cover of Biochemical Journal

Professor Steven Perkins (Professor of Structural Biochemistry, UCL) made the front cover of October’s Biochemical Journal (vol. 476, issue 19) with his latest paper:

Crystal structure of zinc-α2-glycoprotein in complex with a fatty acid reveals multiple different modes of protein-lipid binding

A joint project with UCL Medicine and the University of Bedfordshire, it is a combination of work done on Professor Perkins’ new analytical ultracentrifuge, protein crystallography and recombinant proteins.

Citation: Lau, A. M., Zahid, H., Gor, J., Perkins, S. J., Coker, A. R. & McDermott, L. C. (2019) Crystal structure of zinc-α2-glycoprotein in complex with a fatty acid reveals multiple different modes of lipid binding. Biochem. J. 476, 2815-2834.
Posted by ubcg49z in News, Publications, Uncategorised

Professor Bart Hoogenboom’s group publish paper in ACS Nano

Professor Bart Hoogenboom’s research group published a paper titled ‘Quantification of Biomolecular Dynamics inside Real and Synthetic Nuclear Pore Complexes using Time-Resolved Atomic Force Microscopy’ in ACS Nano in June 2019.

The full paper is available here.

Posted by ublmcr01 in News, Publications, Uncategorised

Studying protein conformation using a new cyclic ion mobility mass spectrometry (cIMMS) device

Dr Konstantinos Thalassinos

We are the first group to publish a paper on how a new cyclic ion-mobility mass-spectrometry (cIMMS) device, manufactured by Waters, can be used to probe protein structure and dynamics. In particular, the tandem ion mobility capabilities of the instrument allow us to probe in very fine detail protein unfolding pathways and for the first time to do so for co-existing and interconverting conformers. We are now using this technology to study proteins involved in protein misfolding diseases such as human amyloid islet polypeptide.

The paper is:
Eldrid, C.; Ujma, J.; Kalfas, S.; Tomczyk, N.; Giles, K.; Morris, M.; Thalassinos, K. Gas Phase Stability of Protein Ions in a Cyclic Ion Mobility Spectrometry Traveling Wave Device. Anal. Chem. 2019, 91 (12), 7554–7561 https://doi.org/10.1021/acs.analchem.8b05641

A video where I and other people in the field describe the cIMMS technology:
https://vimeo.com/318178536/005117727e

The link to the Waters site:
https://www.waters.com/waters/en_US/SELECT-SERIES-Cyclic-IMS-ion-mobility-mass-spectrometer/nav.htm?cid=135021297&locale=en_PT

 

Posted by ublmcr01 in Lab news, News, Publications, Uncategorised