Day: 23 November 2021

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

Nobel Prize in Chemistry for Bio-Inspired Catalysts

The Nobel Prize in Chemistry in 2021 has been awarded to German Benjamin List and British David MacMillan.
Prof Stefan Howorka from the ISMB at UCL Chemistry explains: ‘The two researchers have developed a new class of catalysts that are inspired by Nature. Enzymes are widely used in biology as they initiate and specifically control biochemical reactions to achieve the desired stereochemistry while limiting the creation of undesirable by-products. Reconstructing these catalytic functions with smaller and cheaper synthetic units is of considerable scientific and industrial interest. Ideally, synthetic catalysts should also avoid precious metals such as platinum which are not environmentally friendly.
List and MacMillan succeeded independently of each other in developing efficient biomimetic and “green” catalysts. In the late 1990s, List wondered whether amino acids found in the enzymes’ active site would also be able to achieve part of the same catalytic role if added in isolation. As proof-of-principle, List tested the catalytic properties of proline and related compounds in an aldol reaction. The specific question was whether the use of a chiral proline would control the stereochemical outcome of the reaction. Indeed, the chirality of the catalyst controlled which enantiomer of the aldol products was formed.
MacMillan was working in the same field. MacMillan was motivated to develop new catalysts that avoid the widely used metals. Rather, he focused on environmentally harmless and inexpensive organic frameworks that contain -in addition to carbon- oxygen, nitrogen, sulphur or phosphorous. Similar to List, MacMillan also tested chiral versions of his organic catalysts but with a different reaction, the Diels-Alder cycloaddition. The reaction was successful as enantiopure products formed depending on the chirality of the catalysts.
Reflecting the catalysts’ composition and enantioselective control, MacMillan coined the term ‘asymmetric organocatalysis’ This new field has grown dramatically and develops simple, easy-to-manufacture and environmentally friendly catalyst. This has a huge impact in science and industry to produce new pharmaceuticals or molecules that can capture light in solar cells. This year’s Nobel prize and the Nobel prize given in 2018 for ‘the directed evolution of enzymes’ underscore the importance of developing new catalytic tools, Prof Howorka concludes.

References:
J. Am. Chem. Soc. 2000, 122, 2395-2396; J. Am. Chem. Soc. 2000, 122, 4243-4244

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