Biology subjects

Thayer, K. M.

Publications and source records attributed to Thayer, K. M..

7 recordsLinked to original sources

bioRxiv2026

Anticodon nucleotide modifications affect translational tuning by the ribosomal CAR surface

Nucleotide modifications of the tRNA anticodon can affect protein translation fidelity and speed. Chemical modifications of the anticodon nucleotide 34 are regulated under cellular stress and associated with several translational defects and pathologies. Here, we investigate how these modifications influence A-site codon recognition interactions and their coupling to the CAR site that lies adjacent to nucleotide 34 in the ribosome. The conserved three-residue CAR interface hydrogen bonds in a sequence-dependent manner to the mRNA +1 codon 3-adjacent to the A-site codon and is implicated in tuning translational speed. The C of CAR is pi-stacked with the nucleotide 34 of the A site tRNA anticodon. The A site and the CAR site influence each others hydrogen bonding and stacking interactions, and these codon-adjacency effects potentially provide a layer of regulation affecting translational fidelity and kinetics. Through molecular dynamics simulations of a subsystem of a translocating ribosome IRES-model, we observed that nucleotide 34 modifications affect the hydrogen bonding and stacking interactions at the A site and CAR site as well as CARs influence on the A site interactions. Integrating these results with gene sequence and ribosome profiling analyses, we propose that nucleotide 34 modifications help modulate CARs sequence-dependent tuning of translation in response to cellular stress. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/740946v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1971867org.highwire.dtl.DTLVardef@c31822org.highwire.dtl.DTLVardef@6597f6org.highwire.dtl.DTLVardef@1dbd747_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2025

StACKER, A TOOL FOR SYSTEMS LEVEL ANALYSIS OF BASE STACKING IN NUCLEOTIDE-RICH STRUCTURES

Nucleic acid macromolecules can undergo significant structural changes--often mediated by nucleotide modification or molecular interactions. These alterations can be critical to their function. An ongoing challenge is to develop useful tools to quantify system-wide changes in nucleic acid-rich structures. We introduce StACKER, a robust Python package for observing conformational changes in a nucleic acid structure through its pi-stacking of base rings. StACKER creates System Stacking Fingerprints (SSFs) which highlight the landscape of pi-stacking throughout a molecule and can be used to show widespread conformational adjustments. Additionally, StACKERs Pairwise Stacking Fingerprint (PSF) can further characterize pi-stacking in a select residue pair, showing how the effects of localized residue changes spread through a nucleic acid system. We apply StACKER to molecular dynamics (MD) simulations of a subsystem of the ribosome to reveal that alternative codons at the ribosome A-site and 3 adjacent +1 codon position induce allosteric structure changes in the decoding center neighborhood, leading to a toggle between two conformational states. Through previous analysis of ribosome profiling data, we link these states to an observed fast/slow translation phenotype (Sun et al. 2024). We benchmark StACKER alongside other lenses for observing allostery to demonstrate its use in assessing allosteric shifts and its relevance to future structure-to-function studies.

bioinformatics↗

bioRxiv2024

Structural and mechanistic diversity in p53-mediated regulation of organismal longevity across taxonomical orders

The accumulation of senescent cells induces several aging phenotypes, and the p53 tumor suppressor protein regulates one of the two known cellular senescence pathways. p53s regulation of senescence is however not clear. For example, p53 deficiency in some mice has been shown to rescue premature aging while others display significant aging phenotype when p53-deficient. This study seeks to elucidate, structurally and mechanistically, p53s roles in longevity. Through a relative evolutionary scoring (RES) algorithm, we quantify the level of evolutionary change in the residues of p53 across organisms of varying average lifespans in six taxonomic orders. Secondly, we used PEPPI to assess the likelihood of interaction between p53-or p53-linked proteins-and known senescence-regulating proteins across organisms in the orders Primates and Perciformes. Our RES algorithm found variations in the alignments within and across orders, suggesting that mechanisms of p53-mediated regulation of longevity may vary. PEPPI results suggest that longer-lived species may have evolved to regulate induction and inhibition of cellular senescence better than their shorter-lived counterparts. With experimental verification, these predictions could help elucidate the mechanisms of p53-mediated cellular senescence, ultimately clarifying our understanding of p53s connection to aging in a multiple-species context. Author summaryThe p53 tumor suppressor protein protects our genome from cancers by repairing DNA damage, regulating cell death and/or pushing cells to a state where they become permanently unable to divide (known as cellular senescence). An accumulation of senescent cells produces various molecular features of aging in both mouse and human cellular models-thus linking p53 to the aging process. However, the molecular mechanism by which p53 regulates aging and its structural implications on this regulation are not clear. In this study, we assessed quantitatively the evolutionary differences in p53 sequences of organisms across several taxonomical orders to determine if there is a relationship between average lifespan and sequence evolution. In addition, we used a protein-protein interaction tool to assess the likelihood of interaction between p53, or p53-associated protein, and various senescence-associated proteins across organisms of various lifespans in two taxonomic orders: Primates and Perciformes. An elucidation of p53 structural difference and mechanistic proteomic network linked to p53 regulation of cellular senescence could advance therapeutics targeting abnormal aging.

bioinformatics↗

bioRxiv2024

GNN codon adjacency regulates protein translation

The central dogma treats the ribosome as a molecular machine that reads one mRNA codon at a time as it adds each amino acid to its growing peptide chain. However, this and previous studies suggest that ribosomes actually perceive pairs of adjacent codons as they take three-nucleotide steps along the mRNA. We examined GNN codons which we find are surprisingly overrepresented in eukaryote protein-coding open reading frames (ORFs), especially immediately after NNU codons. Ribosome profiling experiments in yeast revealed that ribosomes with NNU at their aminoacyl (A) site have particularly elevated densities when NNU is immediately followed (3) by a GNN codon, indicating slower mRNA threading of the NNU codon from the ribosomes A to peptidyl (P) sites. Moreover, if the assessment was limited to ribosomes that have only recently arrived at the next codon, by examining 21-nucleotide ribosome footprints (21-nt RFPs), elevated densities were observed for multiple codon classes when followed by GNN. This striking translation slowdown at adjacent 5-NNN GNN codon pairs is likely mediated in part by the ribosomes CAR surface which acts as an extension of the A-site tRNA anticodon during ribosome translocation and interacts through hydrogen bonding and pi stacking with the GNN codon. The functional consequences of 5-NNN GNN codon adjacency are expected to influence the evolution of protein coding sequences. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/583757v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@d29087org.highwire.dtl.DTLVardef@e8f7dorg.highwire.dtl.DTLVardef@1ee5792org.highwire.dtl.DTLVardef@b750fb_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

molecular biology↗

bioRxiv2020

Arginine Methylation Regulates Ribosome CAR Function

The ribosome CAR interaction surface is hypothesized to provide a layer of translation regulation through hydrogen-bonding to the +1 mRNA codon that is next to enter the ribosome A site during translocation. The CAR surface consists of three residues, 16S/18S rRNA C1054, A1196 (E. coli 16S numbering), and R146 of yeast ribosomal protein Rps3. R146 can be methylated by the Sfm1 methyltransferase which is downregulated in stressed cells. Through molecular dynamics analysis, we show here that methylation of R146 compromises the integrity of CAR by reducing the pi stacking of the R146 guanidinium group with A1196, leading to reduced CAR hydrogen-bonding with the +1 codon. We propose that ribosomes assembled under stressed conditions have unmethylated R146, resulting in elevated CAR/+1 codon interactions, which tunes translation levels in response to the altered cellular context.

biophysics↗

bioRxiv2020

GCN sensitive protein translation in yeast

Levels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons--the ramp region--of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN)n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.

molecular biology↗

bioRxiv2020

A Ribosome Interaction Surface Sensitive to mRNA GCN Periodicity

GCN codons are over-represented in initial codons of ORFs of prokaryote and eukaryote mRNAs. We describe a ribosome rRNA-protein surface that interacts with an mRNA GCN codon when next-in-line for the ribosome A site. The interaction surface is comprised of the edges of two stacked rRNA bases: the Watson-Crick edge of 16S/18S rRNA C1054 and adjacent Hoogsteen edge of A1196 (Escherichia coli 16S rRNA numbering). Also part of the interaction surface, the planar guanidinium group of a conserved Arginine (R146 of yeast ribosomal protein Rps3) is stacked adjacent to A1196. On its other side, the interaction surface is anchored to the ribosome A site through base stacking of C1054 with the wobble anticodon base of the A-site tRNA. Using Molecular Dynamics simulations of a 495-residue subsystem of translocating ribosomes, we observe base pairing of C1054 to nucleotide G at position 1 of the next-in-line codon, consistent with previous cryo-EM observations, and hydrogen bonding of A1196 and R146 to C at position 2. Hydrogen bonding to both of these codon positions is significantly weakened when C at position 2 is changed to G, A or U. These sequence-sensitive mRNA-ribosome interactions at the C1054-A1196-R146 (CAR) surface potentially contribute to GCN-mediated regulation of protein translation.

biophysics↗