Biology subjects

Santangelo, T. J.

Publications and source records attributed to Santangelo, T. J..

3 recordsLinked to original sources

bioRxiv2025

The Evolutionary Landscape of tRNA Modifications in Archaea: Insights from High-Throughput Sequencing

Transfer RNA (tRNA) modifications are essential for structural integrity, decoding fidelity, and stress adaptation, yet their evolutionary dynamics remain poorly understood. Here, we apply Ordered Two-Template Relay sequencing (OTTR-seq) to comprehensively profile tRNA modifications across nine archaeal species spanning diverse ecological niches. We uncover coordinated and mutually exclusive methylation at acceptor stem positions 6 and 67 in hyperthermophiles, as well as clade-specific co-modification at positions 10 and 26, which are typically known for their importance as tRNA modification anti-determinants. Comparative analyses also reveal lineage-specific divergence in the domain architectures of tRNA methyltransferases, including Trm14, Trm10, Trm11, and Trm1. We further refine known identity elements such as the G10oU25 pairing, and highlight novel structural contexts that facilitate or prevent modification. These findings exemplify the co-evolution of tRNAs and their modifying enzymes, providing new insights into how archaea may fine-tune translation in extreme environments. The scope of these data and comparative analyses establish a multispecies framework for future biochemical, mechanistic, and predictive modeling efforts.

evolutionary biology↗

bioRxiv2024

A sequence-specific RNA acetylation catalyst

N4-acetylcytidine (ac4C) is a ubiquitous RNA modification incorporated by cytidine acetyltransferase enzymes. Here, we report the biochemical characterization of Thermococcus kodakarensis Nat10 (TkNat10), an RNA acetyltransferase involved in archaeal thermotolerance. We demonstrate that TkNat10s catalytic activity is critical for T. kodakarensis fitness at elevated temperatures. Unlike eukaryotic homologs, TkNat10 exhibits robust stand-alone activity, modifying diverse RNA substrates in a temperature, ATP, and acetyl-CoA-dependent manner. Transcriptome-wide analysis reveals TkNat10 preferentially modifies unstructured RNAs containing a 5-CCG-3 consensus sequence. Using a high-throughput mutagenesis approach, we define sequence and structural determinants of TkNat10 substrate recognition. We find TkNat10 can be engineered to use non-native acyl-CoA donors, providing insight into its cofactor specificity. Finally, we demonstrate TkNat10s utility for site-specific acetylation of RNA oligonucleotides, enabling analysis of ac4C-dependent RNA-protein interactions. Our findings establish a framework for understanding archaeal RNA acetylation and a new tool for studying the functional consequences of ac4C in diverse RNA contexts.

biochemistry↗

bioRxiv2023

Structural basis of archaeal FttA-dependent transcription termination

The ribonuclease FttA mediates factor-dependent transcription termination in archaea1-3. Here, we report the structure of a Thermococcus kodakarensis transcription pre-termination complex comprising FttA, Spt4, Spt5, and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic center and that enables endonucleolytic cleavage of RNA by FttA, followed by 5[->]3 exonucleolytic cleavage of RNA by FttA and concomitant 5[->]3 translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination. The structure further reveals that Spt5 bridges FttA and the TEC, explaining how Spt5 stimulates FttA-dependent termination. The results reveal functional analogy between bacterial and archaeal factor-dependent termination, reveal functional homology between archaeal and eukaryotic factor-dependent termination, and reveal fundamental mechanistic similarities in factor-dependent termination in the three domains of life: bacterial, archaeal, and eukaryotic. One sentence summaryCryo-EM reveals the structure of the archaeal FttA pre-termination complex

molecular biology↗