RESUMEN
The 22 mitochondrial and â¼45 cytosolic tRNAs in human cells contain several dozen different post-transcriptional modified nucleotides such that each carries a unique constellation that complements its function. Many tRNA modifications are linked to altered gene expression, and deficiencies due to mutations in tRNA modification enzymes (TMEs) are responsible for numerous diseases. Easily accessible methods to detect tRNA hypomodifications can facilitate progress in advancing such molecular studies. Our laboratory developed a northern blot method that can quantify relative levels of base modifications on multiple specific tRNAs â¼10 yr ago, which has been used to characterize four different TME deficiencies and is likely further extendable. The assay method depends on differential annealing efficiency of a DNA-oligo probe to the modified versus unmodified tRNA. The signal of this probe is then normalized by a second probe elsewhere on the same tRNA. This positive hybridization in the absence of modification (PHAM) assay has proven useful for i6A37, t6A37, m3C32, and m2,2G26 in multiple laboratories. Yet, over the years we have observed idiosyncratic inconsistency and variability in the assay. Here we document these for some tRNAs and probes and illustrate principles and practices for improved reliability and uniformity in performance. We provide an overview of the method and illustrate benefits of the improved conditions. This is followed by data that demonstrate quantitative validation of PHAM using a TME deletion control, and that nearby modifications can falsely alter the calculated apparent modification efficiency. Finally, we include a calculator tool for matching probe and hybridization conditions.
Asunto(s)
Northern Blotting/métodos , ARN de Transferencia/química , Células HEK293 , Humanos , Metilación , Procesamiento Postranscripcional del ARN , ARN de Transferencia/metabolismo , LevadurasRESUMEN
N6-threonyl-carbamoyl adenosine (t6A) is a universal tRNA modification found at position 37, next to the anticodon, in almost all tRNAs decoding ANN codons (where N = A, U, G, or C). t6A stabilizes the codon-anticodon interaction and hence promotes translation fidelity. The first step of the biosynthesis of t6A, the production of threonyl-carbamoyl adenylate (TC-AMP), is catalyzed by the Sua5/TsaC family of enzymes. While TsaC is a single domain protein, Sua5 enzymes are composed of the TsaC-like domain, a linker and an extra domain called SUA5 of unknown function. In the present study, we report structure-function analysis of Pyrococcus abyssi Sua5 (Pa-Sua5). Crystallographic data revealed binding sites for bicarbonate substrate and pyrophosphate product. The linker of Pa-Sua5 forms a loop structure that folds into the active site gorge and closes it. Using structure-guided mutational analysis, we established that the conserved sequence motifs in the linker and the domain-domain interface are essential for the function of Pa-Sua5. We propose that the linker participates actively in the biosynthesis of TC-AMP by binding to ATP/PPi and by stabilizing the N-carboxy-l-threonine intermediate. Hence, TsaC orthologs which lack such a linker and SUA5 domain use a different mechanism for TC-AMP synthesis.