Roman Tuma

University of Leeds · Astbury Centre for Structural Molecular Biology (ACSMB)

Significance Accurate RNA folding is essential for virus replication. Rotaviruses are viruses infecting humans and animals. Rotavirus genome comprises 11 distinct RNAs, and successful replication requires the incorporation of all 11 RNAs into a virion. The RNA chaperone NSP2 binds viral transcripts, regulating their interactions with each other. NS...

Rotavirus genomes are distributed between 11 distinct RNA segments, all of which are essential for virus replication. Stoichiometric genome segment selection and assembly is achieved through a series of sequence-specific, intersegment RNA-RNA interactions that are facilitated by the rotavirus RNA chaperone protein NSP2. The C-terminal region (CTR)...

Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein co...

Hydrogen/deuterium exchange monitored by mass spectrometry is a promising technique for rapidly fingerprinting structural and dynamical properties of proteins. The time-dependent change in the mass of any fragment of the polypeptide chain depends uniquely on the rate of exchange of its amide hydrogens, but determining the latter from the former is...

Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry (MS) is a promising technique for rapidly fingerprinting structural and dynamical properties of proteins. The time dependent change in mass of any fragment of the polypeptide chain depends uniquely on the rate of exchange of its amide hydrogens but determining the latter from the form...

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques...

Primary single molecule fluorescence data file for use with the Python script.

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To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA–RNA interactions between single-stranded RNA segment precursors....

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors....

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon – SecY MK EG – resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolys...

Formation of the Hepatitis B (HBV) nucleocapsid (NC) is an essential step in the viral lifecycle but its assembly is not fully understood. We report the discovery of sequencespecific interactions between the viral pre-genome and HBV core protein (Cp) that play roles in defining the NC assembly pathway. Using RNA SELEX and bioinformatics we identifi...

Long RNA molecules are at the core of gene regulation across all kingdoms of life, whilst also serving as genomes in RNA viruses. Few studies have addressed the basic physical properties of long single-stranded RNAs. Long RNAs with non-repeating sequences usually adopt highly ramified secondary structures and are better described as branched polyme...

Chromatin-remodeling ATPases modulate histones-DNA interactions within nucleosomes and regulate transcription. At the heart of remodeling, ATPase is a helicase-like motor flanked by a variety of conserved targeting domains. CHD4 is the core subunit of the nucleosome remodeling and deacetylase complex NuRD and harbors tandem plant homeo finger (tPHD...

Using RNA-coat protein crosslinking we have shown that the principal RNA recognition surface on the interior of infectious MS2 virions overlaps with the known peptides that bind the high affinity translational operator, TR, within the phage genome. The data also reveal the sequences of genomic fragments in contact with the coat protein shell. These...

In bacterial RNA polymerase (RNAP), the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNAP, we are able to show that functions of these regions a...

In bacterial RNA polymerase, the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNA polymerase, we are able to show that functions of these region...

Reoviruses are important human, animal and plant pathogens having 10-12 segments of double-stranded genomic RNA. The mechanisms controlling the assortment and packaging of genomic segments in these viruses, remain poorly understood. RNA-protein and RNA-RNA interactions between viral genomic segment precursors have been implicated in the process. Wh...

Significance Oligomers formed en route to amyloid fibrils are thought to be the perpetrators of toxicity in many amyloid disorders. How amyloid fibrils contribute to disease, however, is less clear. Here, using β 2 -micoglobulin (β 2 m) as a model system, we show that the stability of amyloid fibrils is highly pH-dependent, with mild acidification...

Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using β 2-microglobulin (β 2 m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused b...

Significance Single-stranded RNA viruses self-assemble protective protein containers around their cognate genomes rapidly and efficiently at low concentrations. RNA encapsidation in vivo occurs preferentially with the cognate genome, in contrast to many in vitro reassembly experiments. We describe in molecular detail how this specificity and effici...

The biological function of large macromolecular assemblies depends on their structure and their dynamics over a broad range of timescales; for this reason, it is a significant challenge to investigate these assemblies using conventional experimental techniques. One of the most promising experimental techniques is hydrogen-deuterium exchange detecte...

Chlorosomes are light-harvesting complexes found in photosynthetic bacteria belonging to three diverse phyla: Chlorobi, Chloroflexi and Acidobacteria. They are composed of bacteriochlorophylls with minor contributions from proteins, lipids, carotenoids and quinones. Proteins are confined to the surface of the chlorosome while most bacteriochlorophy...

Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ70 or σ54, that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ54 version controls gene expression in response to stress, often mediating pathogenicity....

TALE (transcription activator-like effector) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. In the present study, we have systematically assessed...

Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocatio...

The formation of a protective protein container is an essential step in the life-cycle of most viruses. In the case of single-stranded (ss)RNA viruses, this step occurs in parallel with genome packaging in a co-assembly process. Previously, it had been thought that this process can be explained entirely by electrostatics. Inspired by recent single-...

Long RNAs often exist as multiple conformers in equilibrium. For the genomes of single-stranded RNA viruses, one of these conformers must include a compacted state allowing the RNA to be confined within the virion. We have used single molecule fluorescence correlation spectroscopy to monitor the conformations of viral genomes and sub-fragments in t...

Chlorosomes are large light-harvesting complexes found in three phyla of anoxygenic photosynthetic bacteria. Chlorosomes are primarily composed of self-assembling pigment aggregates. In addition to the main pigment, bacteriochlorophyll c, d or e, chlorosomes also contain variable amounts of carotenoids. Here, we use X-ray scattering and electron cr...

Chlorosomes are light harvesting complexes, interior of which is composed of bacteriochlorophylls that are assembled into lamellar aggregates. In this work, short-range structural parameters of aggregated bacteriochlorophyll-c were explored. The knowledge of bonds involved in aggregation together with other experimental results provides constraints...

P4 proteins are hexameric RNA packaging ATPases of dsRNA bacteriophages of the Cystoviridae family. P4 hexamers are integral part of the inner polymerase core and play several essential roles in the virus replication cycle. P4 proteins are structurally related to the hexameric helicases and translocases of superfamily 4 (SF4) and other RecA-like AT...

Genome packaging is an essential step in virus replication and a potential drug target. Single-stranded RNA viruses have been thought to encapsidate their genomes by gradual co-assembly with capsid subunits. In contrast, using a single molecule fluorescence assay to monitor RNA conformation and virus assembly in real time, with two viruses from dif...

CHD4, the core subunit of the Nucleosome Remodelling and Deacetylase (NuRD) complex, is a chromatin remodelling ATPase that, in addition to a helicase domain, harbors tandem plant homeo finger and chromo domains. By using a panel of domain constructs we dissect their roles and demonstrate that DNA binding, histone binding and ATPase activities are...

Background / Purpose: Transcription and translation are fundamental cellular processes that mediate conversion of the DNA encoded information into proteins. In bacteria, transcription is the key step in gene expression regulation and is mediated by a core RNA polymerase complex (E) comprising 2 alpha, 2 beta, beta' and omega subunits and the prom...

The unwinding of RNA helices and the disruption of RNA-protein complexes are critical steps of cellular metabolism that are carried out by ubiquitous NTP-dependent enzymes named RNA helicases. Here, we review the structures, mechanisms, and biochemical properties of two RNA helicases known to adopt a homo-hexameric ring architecture: the P4 packagi...

Chlorosomes from green photosynthetic bacteria are large photosynthetic antennae containing self-assembling aggregates of bacteriochlorophyll c, d, or e. The pigments within chlorosomes are organized in curved lamellar structures. Aggregates with similar optical properties can be prepared in vitro, both in polar as well as non-polar solvents. In or...

Assembly of immature retroviral particles is a complex process involving interactions of several specific domains of the Gag polyprotein localized mainly within capsid protein (CA), spacer peptide (SP), and nucleocapsid protein (NC). In the present work we focus on the contribution of NC to the oligomerization of CA leading to assembly of Mason-Pfi...

The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from C. aurantiacus was investigated by using a combination of cryo-electron microsc...

Optical tweezers perform in singulo experiments on biological reactions that occur stochastically, often through multiple pathways. Characterization of single molecule trajectories allows determination of conformational distributions and detection of intermediates. However, this approach requires repeating the measurements tens or hundreds of times...

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodispers...

Using real-time re-programmable signal processing we connect acousto-optic steering and back-focal-plane interferometric position detection in optical tweezers to create a fast feedback controlled instrument. When trapping 3 µm latex beads in water we find that proportional-gain position-clamping increases the effective lateral trap stiffness ~13-f...

We present lamellar self-assembly of cationic poly(L-histidine) (PLH) stoichiometrically complexed with an anionic surfactant, dodecyl benzenesulfonic acid (DBSA), which allows a stabilized conformation reminiscent of polyproline type II (PPII) left-handed helices. Such a conformation has no intrapeptide hydrogen bonds, and it has previously been f...

Chlorosomes are light-harvesting complexes of green photosynthetic bacteria. Chlorosomes contain bacteriochlorophyll (BChl) c, d, or e aggregates that exhibit strong excitonic coupling. The short-range order, which is responsible for the coupling, has been proposed to be augmented by pigment arrangement into undulated lamellar structures with spaci...

The P4 protein of bacteriophage φ12 is a hexameric molecular motor closely related to superfamily 4 helicases. P4 converts chemical energy from ATP hydrolysis into mechanical work, to translocate single-stranded RNA into a viral capsid. The molecular basis of mechanochemical coupling, i.e. how small ∼1 Å changes in the ATP-binding site are amplifie...

Recent advances in protein mass spectrometry (MS) have enabled determinations of hydrogen deuterium exchange (HDX) in large macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed pro...

The bacteriophages of the Cystoviridae family package their single stranded RNA genomic precursors into empty capsid (procapsids) using a hexameric packaging ATPase motor (P4). This molecular motor shares sequence and structural similarity with RecA-like hexameric helicases. A concerted structural, mutational and kinetic analysis helped to define t...

Murray Valley encephalitis virus (MVEV), a mosquito-borne flavivirus endemic to Australia, is closely related to Japanese encephalitis virus and West Nile virus. Nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains, whose activity is central to flavivirus replication and is therefore a possi...

A label- and immobilization-free approach to detecting the reversible formation of complexes between nucleic acids and proteins at the single-molecule level is described. The voltage-driven translocation of individual oligoribonucleotides through a nanoscale protein pore is observed by single-channel current recordings. The oligoribonucleotide 5'-C...

Optically trapped microshperes can be manipulated by steering the trap beam, while the object position is measured with sub-nanometer resolution. A fast steering system is required to create feedback loop for measurements at a constant force or to increase position detection precision by trap stiffening. Using a real-time re-programmable digital si...

Linear molecular motors translocate along polymeric tracks using discrete steps. The step length is usually measured using constant-force single molecule experiments in which the polymer is tethered to a force-clamped microsphere. During the enzymatic cycle the motor shortens the tether contour length. Experimental conditions influence the achievab...

Chlorosomes, the main antenna complexes of green photosynthetic bacteria, were isolated from null mutants of Chlorobium tepidum, each of which lacked one enzyme involved in the biosynthesis of carotenoids. The effects of the altered carotenoid composition on the structure of the chlorosomes were studied by means of x-ray scattering and electron cry...

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent relea...

Chlorosomes are the main light harvesting complexes of green photosynthetic bacteria. Recently, a lamellar model was proposed for the arrangement of pigment aggregates in Chlorobium tepidum chlorosomes, which contain bacteriochlorophyll (BChl) c as the main pigment. Here we demonstrate that the lamellar organization is also found in chlorosomes fro...

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