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Optimizing conditions for cleavage of pre‐tRNA‐Gln(CAA) by Haloferax volcanii tRNA intron endonuclease
Author(s) -
Tellings Kelsey,
Salva Corinne,
GinnPease Margaret E.,
Hemmingsen Jens M.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.884.3
Subject(s) - transfer rna , haloferax volcanii , intron , endonuclease , biochemistry , nucleotide , cleavage (geology) , rna , chemistry , enzyme , biology , microbiology and biotechnology , archaea , gene , paleontology , fracture (geology)
In the halophilic archaeon Haloferax volcanii , tRNA intron endonuclease is known to cleave precursor tRNAs at a specific bulge‐helix‐bulge (BHB) motif. The enzyme cleaves pre‐tRNA‐Trp into a 103 nucleotide intron and two exons that are subsequently spliced at position 37/38. To explore the substrate specificity of the enzyme, pre‐tRNA‐Gln(CCA) has been subjected to cleavage by tRNA intron endonuclease under various conditions. Pre‐tRNA‐Gln(CCA) contains a shorter intron of 31 nucleotides, but can still adopt a BHB motif. Optimal cleavage conditions appear to be 40 mM Tris‐HCl pH 7.4, 2% glycerol, 20 mM MgCl 2 and 2 M KCl. The enzyme activity is not sensitive to moderate pH changes or MgCl 2 concentration between 5 mM and 50 mM. Ionic strength is important with increasing activity observed at higher KCl concentrations from 200 mM to 2M KCl and no cleavage observed below 200 mM KCl. These studies will allow for additional exploration of substrate specificity including sequence variation in the RNA substrate. This work will lead to a better understanding of tRNA processing in archaea.