Site-directed in vitro replacement of nucleosides in the anticodon loop of tRNA: application to the study of structural requirements for queuine insertase activity.

Abstract

We have investigated the specificity of the enzymes Q‐insertase and mannosyl‐Q transferase that replace the guanosine at position 34 (wobble base) in the anticodon of several tRNAs by Q or mannosyl‐Q derivatives. We have restructured in vitro the normal anticodon of yeast tRNA‐Asp‐GUC, yeast tRNAArgICG and yeast tRNALeuUAG. With yeast tRNA‐Asp‐GUC, we have replaced one or several nucleotides in the vicinity of G34 by one of the four canonical nucleotides or by pseudouridylic acid; we have also constructed a tRNAAsp with eight bases instead of seven in the anticodon loop. With yeast tRNAArgICG and yeast tRNALeuUAG, we have replaced their anticodon by the trinucleotide GUC, coding for aspartic acid. The chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and after 72 h the amount of Q34 and mannosyl‐Q34 incorporated was measured. Our results show that the U33G34U35 sequence, within an anticodon loop of seven bases in chimerical yeast tRNA‐Asp‐GUC, tRNAArgGUC or tRNALeuGUC, is the main determinant for Q‐insertase activity at position 34; the rest of the tRNA sequence has only a slight influence. For mannosyl‐Q transferase, however, a much broader structural feature of the tRNA than just the U33G34U35 sequence is important for the efficiency of Q34 transformation into mannosyl‐Q34.

C to U editing and modifications during the maturation of the mitochondrial tRNAASPin marsupials

“…This is in accordance with the observation that tRNAs which have a U at the position immediately 5' of anticodons with the general sequence GUN carry queuosine residues at the first position of the anticodon (28). In E.coli (29) and in Xenopus laevis oocytes (30), the enzyme that incorporates queuine into tRNAs requires the target sequence UGU (bold letters represent the two first positions of the anticodon) in order to replace the guanine by queuine. Therefore, guanine is likely to From a third tRNA preparation, the sequence of a band of intermediate molecular weight was determined.…”

Overexpression of initiator methionine tRNA leads to global reprogramming of tRNA expression and increased proliferation in human epithelial cells

“…Furthermore, because tRNAs are highly abundant, overexpression even by twofold would require an additional transcription of ∼1,000,000 molecules of tRNA i Met per cell (Pavon-Eternod et al 2009). In the past, exogenous tRNA has been introduced into cells either as DNA or directly as RNA (Carbon et al 1983;Buvoli et al 2000). We tried three different approaches to increase tRNA i Met levels in two human cell lines: transient transfection with tRNA transcripts, transient transfection with a DNA vector containing the tRNA gene, and stable transfection with a DNA vector containing the tRNA gene.…”

A dual fluorescent reporter for the investigation of methionine mistranslation in live cells

“…We monitored mistranslation changes upon transfection of the transcripts of these seven tRNA Glu s. tRNA Glu isodecoders were synthesized in vitro with the proper 5 ′ ends generated by RNase P cleavage, then directly transfected to stable GAA and GAG cells, as done previously (Carbon et al 1983;Buvoli et al 2000;Kohrer et al 2001;Geslain and Pan 2010). The mCherry level was then analyzed by flow cytometry after 36 h. Direct transfection of tRNA transcript at the same amount was done to avoid any potential differences in RNA polymerase III transcription due to sequence differences of the isodecoders.…”