Biology subjects

Jackman, J. E.

Publications and source records attributed to Jackman, J. E..

7 recordsLinked to original sources

bioRxiv2025

Molecular basis of tRNA substrate recognition and modification by the atypical SPOUT methyltransferase Trm10

The evolutionarily conserved methyltransferase Trm10 catalyzes N1 methylation of guanosine 9 (G9) in select tRNAs, but the basis of specific substrate recognition and modification has remained unclear. Using an S-adenosyl-L-methionine analog, we trapped a post-catalytic state of the Trm10-tRNAGly complex for structure determination by cryogenic electron microscopy. Three distinct complexes were captured: two monomeric Trm10-tRNA complexes with distinct tRNA acceptor stem orientations ("closed" and "open"), and a minor dimeric complex with two Trm10s bound to the same tRNA. The monomeric structures identify conserved residues involved in tRNA interactions across a positively charged surface that guide G9 into the catalytic site and stabilize the flipped nucleotide. In the tRNAopen conformation, acceptor stem rotation weakens tRNA-protein contacts, consistent with a product-release state. The dimeric complex, supported by tRNA-dependent protein crosslinking and molecular dynamics (MD) simulations, positions one Trm10 on G9 similarly to the monomeric complexes, while the other contacts distal tRNA regions, suggesting a functional role in promoting functionally critical conformational transition(s). MD simulations also show how Trm10 achieves selective stabilization of G9 over A9 in the binding pocket. Overall, our findings reveal the mechanism of G9-specific tRNA methylation by Trm10 and suggest a unique mechanism of action among RNA-modifying SPOUT methyltransferases.

biochemistry↗

bioRxiv2025

A comparison of Dictyostelium discoideum 3'-5' RNA polymerases reveals a conserved tRNAHis guanylyltransferase residue that plays a dual role in catalysis

The 3-5 RNA polymerase family consists of eukaryotic tRNAHis guanylyltransferase (Thg1) and Thg1 homologs known as Thg1-like proteins (TLPs) that exist in all three domains of life. Thg1 catalyzes an essential reaction adding a G-1 nucleotide to the 5 end of the tRNAHis, forming an identity element for tRNA aminoacylation. All TLPs studied, except Dictyostelium discoideum (Ddi) TLP2, perform in vitro Watson-Crick (WC) dependent addition of multiple nucleotides to repair truncated tRNA. DdiTLP2 has a similar activity to a Thg1 enzyme, adding G-1 to mt-tRNAHis, but shares other biochemical properties with other TLPs, including a restriction to making WC base pairs during this reaction. We identified two regions in DdiTLP2 that lacked residues that are absolutely conserved in other Thg1/TLP enzymes. DdiTLP2 variants in both regions abolish enzymatic activity of DdiTLP2, indicating these regions are important for DdiTLP2 catalysis. Complementary alterations to one of these residues (D150) in DdiThg1 caused an unexpected reversal of this enzymes specificity, with a loss of its ability to incorporate a non-WC base paired G-1 to its physiological substrate, while gaining the ability to add WC base paired G-1 to mt-tRNAHis. These biochemical results, combined with structural models suggest a previously unknown role for D150 in controlling substrate specificity at the adenylation step by providing a checkpoint for correct setup of a WC base pair in the active site. Thg1 also appears to have adapted the role of the ancestral D150 residue for a second function, promoting non-WC nucleotide addition to its eukaryotic tRNAHis substrate.

biochemistry↗

bioRxiv2025

Metal ion requirement for catalysis by 3'-5' RNA polymerases

The two-metal ion mechanism for catalysis of RNA and DNA synthesis by 5'-3' polymerases has been extensively characterized. The 3'-5' polymerase family of enzymes, consisting of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), perform a similar nucleotide addition reaction, but in the reverse direction, adding Watson-Crick base paired NTPs to the 5-ends of RNA substrates, yet the effect of divalent cations beyond magnesium has not been described. Here, we examined the effects of five divalent cations (Mg2+, Mn2+, Co2+, Ni2+ and Ca2+) on templated nucleotide addition activity and kinetics of 5-activation by ATP catalyzed by recombinantly purified, metal-free TLPs from organisms from diverse domains of life. This work revealed that different TLPs exhibit distinct dependencies on the concentration and identity of divalent metal ions that support effective catalysis. The patterns of metal ion usage demonstrated here for TLPs evince features that are characteristic of both canonical 5-3 polymerases and DNA/RNA ligases. Similar to 5'-3' polymerases, some metals were also seen to be mutagenic in the context of TLP catalysis. Furthermore, we provide the first direct evidence that both ATP and the NTP poised for nucleotidyl transfer are present in the active site during the 5-adenylylation. These results provide the first in-depth study of the role of the two-metal ion mechanism in TLP catalysis that was first suggested by structures of these enzymes.

biochemistry↗

bioRxiv2024

Thg1 family 3'-5' RNA polymerases as tools for targeted RNA synthesis

Members of the 3-5 RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), catalyze templated synthesis of RNA in the reverse direction to all other known 5-3 RNA and DNA polymerases. Discovery of enzymes capable of this reaction raised the possibility of exploiting 3-5 polymerases for post-transcriptional incorporation of nucleotides to the 5-end of nucleic acids without ligation, and instead by templated polymerase addition. To date, studies of these enzymes have focused on nucleotide addition to highly structured RNAs, such as tRNA and other non-coding RNA. Consequently, general principles of RNA substrate recognition and nucleotide preferences that might enable broader application of 3-5 polymerases have not been elucidated. Here, we investigated the feasibility of using Thg1 or TLPs for multiple nucleotide incorporation to the 5-end of a short duplex RNA substrate, using a templating RNA oligonucleotide provided in trans to guide 5-end addition of specific sequences. Using optimized assay conditions, we demonstrated a remarkable capacity of certain TLPs to accommodate short RNA substrate-template duplexes of varying lengths with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to 8 bases to 5-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5-end labeling tool.

biochemistry↗

bioRxiv2023

A tRNA-specific function for tRNA methyltransferase Trm10 is associated with a new tRNA quality control mechanism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae a single homolog of the tRNA methyltransferase Trm10 performs m1G9 modification on 13 different tRNAs. Here we provide evidence that the m1G9 modification catalyzed by S. cerevisiae Trm10 plays a biologically important role for one of these tRNA substrates, tRNATrp. Overexpression of tRNATrp (and not any of 38 other elongator tRNAs) rescues growth hypersensitivity of the trm10{Delta} strain in the presence of the antitumor drug 5-fluorouracil (5FU). Mature tRNATrp is depleted in trm10{Delta} cells, and its levels are further decreased upon growth in 5FU, while another Trm10 substrate (tRNAGly) is not affected under these conditions. Thus, m1G9 in S. cerevisiae is another example of a tRNA modification that is present on multiple tRNAs but is only essential for the biological function of one of those species. In addition to the effects of m1G9 on mature tRNATrp, precursor tRNATrp species accumulate in the same strains, an effect that is due to at least two distinct mechanisms. The levels of mature tRNATrp are rescued in the trm10{Delta}met22{Delta} strain, consistent with the known role of Met22 in tRNA quality control, where deletion of met22 causes inhibition of 5-3 exonucleases that catalyze tRNA decay. However, none of the known Met22-associated exonucleases appear to be responsible for decay of hypomodified tRNATrp, based on inability of mutants of each enzyme to rescue growth of the trm10{Delta} strain in the presence of 5FU. Thus, the surveillance of tRNATrp appears to constitute a distinct tRNA quality control pathway in S. cerevisiae.

molecular biology↗

bioRxiv2023

A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α-primase

The mechanism by which polymerase -primase (pol-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of pol-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5'-end facilitates handoff of the primer to pol and increases pol processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of pol and primase affinities for the varied conformations along the chimeric primer/template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by pol-primase.

biochemistry↗

bioRxiv2023

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the 9th nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 14 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) which allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

biochemistry↗