The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

Abstract

Here we describe the many applications of acid urea polyacrylamide gel electrophoresis (acid urea PAGE) followed by Northern blot analysis to studies of tRNAs and aminoacyl-tRNA synthetases. Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Among the examples described are (i) analysis of the effect of mutations in the Escherichia coli initiator tRNA on its aminoacylation and formylation; (ii) evidence of orthogonality of suppressor tRNAs in mammalian cells and yeast; (iii) analysis of aminoacylation specificity of an archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNA(Pro) with cysteine, but does not aminoacylate archaeal tRNA(Cys) with cysteine; (iv) identification and characterization of the AUA-decoding minor tRNA(Ile) in archaea; and (v) evidence that the archaeal minor tRNA(Ile) contains a modified base in the wobble position different from lysidine found in the corresponding eubacterial tRNA.

Figure 1

Role of unique features of E. coli initiator tRNA^fMet in initiation of protein synthesis. (A) Recognition of tRNA^fMet by methionyl-tRNA transformylase (MTF). (B) Separation of the three forms of E. coli initiator tRNA^fMet: tRNA, aminoacyl∼tRNA (aa∼tRNA), and formylaminoacyl∼tRNA (faa∼tRNA) by polyacrylamide gel electrophoresis under acidic conditions (acid urea PAGE) followed by detection of tRNA by Northern hybridization using a [³²P]-labeled DNA oligonucleotide. The markers for aa∼tRNA and faa∼tRNA were generated by in vitro aminoacylation and formylation of tRNA^fMet using purified MetRS and MTF. (C) Acid urea PAGE/Northern analysis of the effect of mutations on aminoacylation and formylation of the mutant tRNAs in vivo. Analysis of tRNA^fMet from E. coli transformants carrying the wild type (W.T.) gene or various mutant tRNA genes as indicated. Control, tRNA isolated from transformants carrying the plasmid vector without any tRNA gene (modified from [6]).

Figure 2

Important role of the amino acid attached to tRNA in formylation and in initiation of protein synthesis in E. coli. (A) E. coli methionyl-tRNA transformylase (MTF) does not recognize Lys∼tRNA^Mi:2 as a substrate [29,30]. (B) Acid urea PAGE/Northern analysis of total RNA isolated from E. coli expressing the Mi:2 mutant elongator tRNA. Lanes 1 and 2, marker tRNA isolated from TG1 cells using a high-copy vector for expression of Mi:2 tRNA; tRNA isolated under acidic conditions before (lane 1) and after deacylation by base-treatment (lane 2). Lanes 3−5, tRNA isolated from CA274 transformants containing an additional plasmid that had no aminoacyl-tRNA synthetase gene (lane 3), the E. coli GlnRS gene (lane 4), or the MetRS gene (lane 5) (modified from [29]).

Figure 3

Orthogonal suppressor tRNAs for site-specific insertion of unnatural amino acids. (A) The orthogonal suppressor tRNA is not recognized by any of the endogenous aminoacyl-tRNA synthetases (grey), but is aminoacylated by the orthogonal aminoacyl-tRNA synthetase (black), which recognizes no other tRNA in the cell and has been modified to use unnatural amino acids. aaRS, aminoacyl-tRNA synthetase; the star indicates the unnatural amino acid of interest. (B) A complete set of orthogonal amber, ochre, and opal suppressor tRNAs for use in mammalian cells [18]. The suppressor tRNAs (hsup2am, hsup2oc and hsup2op) are derived from the E. coli glutamine tRNA. Acid urea PAGE/Northern analysis of total RNA isolated from HEK293T cells, transfected with plasmids carrying genes for the respective suppressor tRNA and E. coli GlnRS. The suppressor tRNAs were visualized using a [³²P]-labeled oligonucleotide complementary to nucleotides 57−72; a [³²P]- labeled oligonucleotide complementary to the human tRNA^Ser was used as internal standard. (C) Orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair for use in yeast [13]. A suppressor tRNA (hM2am) derived from the human initiator tRNA is co-expressed alongside E. coli GlnRS in S. cerevisiae. hM2am is aminoacylated in yeast only in the presence of E. coli GlnRS (top panel; acid urea PAGE/Northern analysis) and is active in suppression (bottom panel). The test for suppression of the met8−1 amber allele was performed in S. cerevisiae HEY301−129 on a selective plate lacking methionine (modified from [13]).

Figure 4

Characterization of M. jannaschii ProRS (ProCysRS). (A) M. jannaschii ProRS (ProCysRS) catalyzes the formation of Cys∼tRNA^Pro but not Cys∼tRNA^Cys [12]. (B) Acid urea PAGE/Northern analysis of the total RNA isolated from M. jannaschii cells and aminoacylated in vitro with proline and cysteine by M. jannaschii ProRS and M. maripaludis CysRS. The blots were probed with [³²P]-labeled oligonucleotides complementary to M. jannaschii tRNA^Pro and tRNA^Cys, as indicated. Bands indicated by 1, 2, and 3 correspond to uncharged tRNA^Pro, Cys∼tRNA^Pro, and Pro∼tRNA^Pro, respectively. Bands indicated by 4 and 5 correspond to uncharged tRNA^Cys and Cys∼tRNA^Cys, respectively. OH⁻, tRNA after deacylation by base-treatment (modified from [12]).

Figure 5

Formation of Cys∼tRNA^Cys in some methanogenic archaea. (A) Two-step conversion of tRNA^Cys to (i) Sep∼tRNA^Cys, catalyzed by O-phosphoseryl-tRNA synthetase (SepRS), and (ii) Cys∼tRNA^Cys, catalyzed by Sep-tRNA:Cys-tRNA synthetase (SepCysS) [49]. (B) Acid urea PAGE/Northern analysis of total M. maripaludis RNA charged with dialyzed M. jannaschii S-100, M. maripaludis CysRS, and M. jannaschii SepRS (Mja 1660/SepRS) in the presence of 20 amino acids (20 aa), phosphoserine (Sep), or a M. jannaschii S-100 cell extract filtrate (Y3). A portion of the sample was deacylated by base-treatment (OH⁻). The blots were probed with [³²P]-labeled oligonucleotides complementary to M. maripaludis tRNA^Cys (modified from [49]).

Figure 6

Identification of the minor isoleucine tRNA in archaea. (A) H. marismortui contains two different CAU anticodon-containing tRNAs, tRNA_12 and tRNA_34, currently annotated as methionine tRNAs. tRNA_12 represents the elongator methionine tRNA (${\mathrm{tRNA}}_{\mathrm{CAU}}^{\mathrm{Met}}$); the minor isoleucine tRNA (${\mathrm{tRNA}}_{\mathrm{XAU}}^{\mathrm{Ile}}$) of H. marismortui is derived from tRNA_34, and carries an unknown modified base (X) in the anticodon responsible for its aminoacylation and decoding specificity [11]. (B) Determination of deacylation rates of tRNA_12 (${\mathrm{tRNA}}_{\mathrm{CAU}}^{\mathrm{Met}}$) and tRNA_34 (${\mathrm{tRNA}}_{\mathrm{XAU}}^{\mathrm{Ile}}$) from H. marismortui. Total RNA was isolated under acidic conditions and subjected to deacylation by base-treatment as described under Experimental Procedures followed by acid urea PAGE/Northern analysis using probes specific for tRNA_12 (top panel) and tRNA_34 (bottom panel), respectively. The calculated half-lives of deacylation are indicated. aa∼tRNA, aminoacyl-tRNA (modified from [11]).

Figure 7

Detection of base modifications. (A) Formation of lysidine in eubacteria. A CAU anticodon-containing tRNA (${\mathrm{tRNA}}_{\mathrm{CAU}}^{\mathrm{Ile}}$) is modified by tRNA^Ile-lysidine synthetase (TilS) generating the minor isoleucine tRNA (${\mathrm{tRNA}}_{\mathrm{LAU}}^{\mathrm{Ile}}$), which carries lysidine at position 34 in the anticodon. (B) In vitro biotinylation of the free α-NH₂ group present in lysidine of ${\mathrm{tRNA}}_{\mathrm{LAU}}^{\mathrm{Ile}}$ from E. coli followed by acid urea PAGE/Northern analysis. A T7 transcript corresponding to ${\mathrm{tRNA}}_{\mathrm{LAU}}^{\mathrm{Ile}}$ was generated as a marker. Lanes 1 and 2, unmodified T7 transcript; lanes 3 and 4, T7 transcript after in vitro modification with lysidine using TilS; lanes 5 and 6, analysis of total RNA from E. coli. A comparison of tRNAs before (lanes 1, 3, 5) and after (lanes 2, 4, 6) in vitro biotinylation is shown (modified from [11]).