Biology subjects

Kouba, T.

Publications and source records attributed to Kouba, T..

4 recordsLinked to original sources

bioRxiv2022

Backtracking of influenza polymerase upon consecutive incorporation of nucleoside analogue T1106 directly observed by high resolution cryo-electron microscopy

The broad-spectrum antiviral pseudobase T705, a fluorinated pyrazinecarboxamide, is incorporated via its triphosphate form into nascent viral RNA by viral RNA-dependent RNA polymerases. Since it mimics guanine or adenine it can act as a mutagen, whereas consecutive incorporation leads to chain termination. Here we examine the structural basis for incorporation and stalling for the case of influenza polymerase, using T1106-TP, the nucleotide form of T1105, the de-fluoro analogue of T705. We used a specially designed template that allows single T1106-MP incorporation at a defined site followed by consecutive T1106-MP incorporation and stalling four nucleotides later, as demonstrated by biochemical analysis. A high-resolution cryoEM structure of influenza A/H7N9 polymerase, stalled after transcribing this template, revealed that the entire product-template duplex has backtracked by five nucleotides. Consequently, the singly incorporated T1106-MP resides at the +1 position and forms an unexpected wobble base-pair with a U in the template. The relative stability of the canonical and wobble T1106:U base-pairs in different contexts is investigated by molecular dynamics simulations. Using a different template and influenza B polymerase we also observe stalling after double incorporation of T1106-MP and structural analysis showed again that backtracking occurs, this time by four nucleotides. These results show that, at least in early elongation, consecutive T1106-MP incorporation into the product destabilises the proximal end of the product-template duplex, promoting irreversible backtracking until a more favourable overall configuration is achieved. These results give new insight into the unusual mechanism of chain termination by pyrazinecarboxamide base analogues.

molecular biology↗

bioRxiv2021

Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity

Lassa virus, which causes annual outbreaks in West Africa with increasing case numbers in recent years, is recognized by the WHO R&D blueprint as a significant threat for public health with high epidemic potential and no effective countermeasures. The viral large (L) protein, which contains the RNA-dependent RNA polymerase, is a key player for transcription of viral mRNA and genome replication. Here we present nine cryo-EM structures of Lassa virus L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved genomic 3 and 5 promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and elongation states, the endonuclease is inhibited by the binding of two distinct L protein peptides in the active site, respectively, whereas in the pre-initiation state, the endonuclease is uninhibited. In the stalled, early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide. In this configuration, the full C-terminal region of the L protein, including the putative cap-binding domain, is highly ordered. The structural data are complemented by in vitro and cell-based studies testing a broad range of L protein mutants to probe functional relevance. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated and will underpin antiviral drug development targeting the arenavirus L protein.

biochemistry↗

bioRxiv2020

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

RNA synthesis is central to life, and RNA polymerase depends on accessory factors for recovery from stalled states and adaption to environmental changes. Here we investigated the mechanism by which a helicase-like factor HelD recycles RNA polymerase. We report a cryo-EM structure of an unprecedented complex between the Mycobacterium smegmatis RNA polymerase and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNA polymerase channels that are responsible for DNA binding and substrate delivery to the active site, thereby locking RNA polymerase in an inactive state. We show that HelD prevents non-specific interactions between RNA polymerase and DNA and dissociates transcription elongation complexes, but does not inhibit RNA polymerase binding to the initiation {sigma} factor. The liberated RNA polymerase can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that removes undesirable nucleic acids from RNA polymerase.

molecular biology↗

bioRxiv2020

Structural and functional characterization of the Severe fever with thrombocytopenia syndrome virus L protein

The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, would be an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron-cryo microscopy and X-ray crystallography, we obtained 3D models covering [~]70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein-RNA interactions and the development of antiviral strategies against this group of emerging human pathogens.

biochemistry↗