First Advisor

Dirk Iwata-Reuyl

Date of Award

Spring 6-2025

Document Type

Thesis

Degree Name

Bachelor of Science (B.S.) in Biochemistry and University Honors

Department

Chemistry

Language

English

Subjects

tRNA, Thermotoga maritima, t6A, hn6A, base modification, radiochemical

DOI

10.15760/honors.1726

Abstract

Transfer RNA (tRNA) is essential for accurate translation of genetic information and plays a role in cellular processes such as lipid aminoacylation and bacterial conjugation. Base modifications help tRNA fold correctly and function properly. An important base modification, N6-threonylcarbamoyladenosine (t6A) occurs at position A37 in the anticodon-stem-loop of tRNA. This modification is crucial for decoding ANN codons. Interestingly, an Archeal organism, Methanocaldococcus jannaschii, can have either t6A and hydroxynorvalylcarbamoyl-6- adenosine (hn6A) modification at the same position on tRNAs. As Tsa proteins catalyze the formation of both t6A and hn6A modifications, this raises the question of whether discrimination between these modifications depends on the tRNA's identity. This study examines both modifications on Thermotoga maritima tRNAThr UGU. All T. maritima genes encoding TsaB, TsaC2, TsaD, and TsaE were expressed in E. coli BL21/C41 cells and were purified to reconstitute the t6A modification pathway in vitro. Radiochemical assays suggests a preference for L-threonine (L-Thr) over L-hydroxynorvaline (L-hnv) in tRNA modification, with a 9.02-fold higher specificity constant (1460 vs. 162 μM⁻¹s⁻¹) driven by L-Thr’s higher binding affinity (Km = 12.1 μM vs. 129.4 μM). While both substrates achieve comparable Vmax values (~0.26 - 0.31 μM/min), L-Thr achieves this efficiency at lower concentrations. The kinetic data of T. maritima tRNAThr UGU suggests t6A is preferred, in contrast to M. jannaschii, where the same tRNA was reported to prefer hn6A modification.

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Persistent Identifier

https://archives.pdx.edu/ds/psu/43833

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