Biology subjects

Schultz, S. K.

Publications and source records attributed to Schultz, S. K..

5 recordsLinked to original sources

bioRxiv2025

The tRNA dihydrouridine synthase DusA has a distinct mechanism in optimizing tRNAs for translation

Dihydrouridine (D) is one of the most highly conserved RNA modifications across all domains of life. D20 within the tRNA D loop is particularly conserved and is formed by DusA in Escherichia coli. However, the mechanisms and cellular functions of DusA and D20 remain poorly understood. Here, we characterize DusAs role in tRNA binding, cofactor oxidation, and modification activity, along with its impact on tRNA maturation and translation. We find that DusA binds tRNA via a two-step mechanism involving a local structural rearrangement and exhibits a higher affinity for previously modified tRNA compared to unmodified tRNA. Unlike the T arm modifying enzymes TrmA and TruB, DusA does not broadly increase cellular aminoacylation for all tRNAs but enhances the charging of specific tRNA species. Despite limited alterations in overall tRNA charging and abundance in cells lacking DusA, DusA selectively improves translation at several specific codons, suggesting a direct contribution for dihydrouridine to the function of certain tRNAs on the ribosome. In conclusion, our findings suggest DusA acts non-redundantly with and complementary to TrmA and TruB in fine-tuning protein synthesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/672980v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1191c4forg.highwire.dtl.DTLVardef@191b43borg.highwire.dtl.DTLVardef@cfceeborg.highwire.dtl.DTLVardef@119c26a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Modifications in the T arm of tRNA globally determine tRNA maturation, function and cellular fitness

All elongator tRNAs harbor 5-methyluridine 54 and pseudouridine 55 in the T arm, which are generated by the enzymes TrmA and TruB, respectively. Escherichia coli TrmA and TruB both act as tRNA chaperones, and strains lacking trmA or truB are outcompeted by wildtype. Here, we investigate how TrmA and TruB contribute to cellular fitness. Deletion of trmA and truB in E. coli causes a global decrease in aminoacylation and alters other tRNA modification such as acp3U47. Whereas overall protein synthesis is not affected in {Delta}trmA and {Delta}truB strains, the translation of a specific subset of codons is significantly impaired, and the expression of many specific proteins is translationally changed. In conclusion, we demonstrate that universal modifications of the tRNA T arm are critical for global tRNA function by enhancing tRNA maturation, tRNA aminoacylation, and translation, thereby improving cellular fitness and explaining trmA and truB conservation.

molecular biology↗

bioRxiv2022

Molecular mechanism of tRNA binding by the Escherichia coli N7 guanosine methyltransferase TrmB

Among the large and diverse collection of tRNA modifications, 7-methylguanosine is frequently found in the tRNA variable loop at position 46. This modification is introduced by the TrmB enzyme, which is conserved in bacteria and eukaryotes. Complementing the report of various phenotypes for different organisms lacking TrmB homologs, we report here hydrogen peroxide sensitivity for the Escherichia coli {Delta}trmB knockout strain. To gain insight into the molecular mechanism of tRNA binding by E. coli TrmB in real-time, we developed a new assay based on introducing a 4-thiouridine modification at position 8 of in vitro transcribed tRNAPhe enabling us to fluorescently labelled this unmodified tRNA. Using rapid kinetic stopped-flow measurements with this fluorescent tRNA, we examined the interaction of wild-type and single substitution variants of TrmB with tRNA. Our results reveal the role of SAM for rapid and stable tRNA binding, the rate-limiting nature of m7G46 catalysis for tRNA release, and the importance of residues R26, T127 and R155 across the entire surface of TrmB for tRNA binding.

biochemistry↗

bioRxiv2021

Multi-Wavelength Analytical Ultracentrifugation of Biopolymer Mixtures and Interactions

Multi-wavelength analytical ultracentrifugation (MW-AUC) is a recent development made possible by new analytical ultracentrifuge optical systems. MW-AUC is suitable for a wide range of applications and biopolymer systems and is poised to become an essential tool to characterize macromolecular interactions. It adds an orthogonal spectral dimension to the traditional hydrodynamic characterization by exploiting unique chromophores in analyte mixtures that may or may not interact. Here we illustrate the utility of MW-AUC for representative classes of challenging biopolymer systems, including interactions between mixtures of different sized proteins with small molecules, mixtures of loaded and empty viral AAV capsids contaminated with free DNA, and mixtures of different proteins, where some have identical hydrodynamic properties, all of which are difficult to resolve with traditional AUC methods. We explain the improvement in resolution and information content obtained by this technique compared to traditional single- or dual-wavelength approaches. We discuss experimental design considerations and limitations of the method, and address the advantages and disadvantages of the two MW optical systems available today, and the differences in data analysis strategies between the two systems.

biophysics↗

bioRxiv2021

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus

Worldwide, [~]250 million people are chronically infected with the hepatitis B virus (HBV) and are at increased risk of cirrhosis and hepatocellular carcinoma. The HBV persists as covalently closed circular DNA (cccDNA), which acts as the template for all HBV mRNA transcripts. Nucleos(t)ide analogs do not directly target the HBV cccDNA and cannot eradicate the HBV. We have discovered a unique structural motif, a G-quadruplex in HBVs pre-core promoter region that is conserved amongst nearly all genotypes, and is central to critical steps in the viral life-cycle including the production of pre-genomic RNA, core and polymerase proteins, and encapsidation. Thus, an increased understanding of the HBV pre-core may lead to the development of novel anti-HBV cccDNA targets. We utilized biophysical methods to characterize the presence of the G-quadruplex, employed assays using a known quadruplex- binding protein (DHX36) to pull-down HBV cccDNA, and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids. This study provides insights into the presence of a G-quadruplex in the HBV pre-core promoter region essential for HBV replication. The evaluation of this critical host-protein interaction site in the HBV cccDNA may ultimately facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

biophysics↗