Article Figures & Data
Figures
- FIG. 1.
Secondary structure of the argU tRNA. The argU10(Ts) mutation, a G-to-A transition at position 1, is indicated. The modified nucleotides found in this tRNA are dihydrouridine (D), 2-thiocytidine (s2C), 5-methylaminomethyluridine (mnn5U), N6-threonine carbamoyl adenosine (t6A), pseudouridine (ψ), and 5-methyluridine (T) (17, 33).
- FIG. 2.
Growth of SF151 transformed with the following plasmids: vector pCL1 (a); the argU+ plasmid pCL2 (b); the argU(Ts) plasmid pCL3 (c); the argU alleles with U20, G20, and C20 (d, e, and f, respectively), each cloned in pCL1; vector pCL1 together with the ArgRS-overproducing plasmid pYA1 (g); the argU alleles with U20, G20, and C20 (h, i, and j, respectively), each together with pYA1; vector pBR322 (k); the argU+ plasmid pDM1 (l); and pBR322 carrying the argU gene with a CCT anticodon (m). Colonies transformed at 30°C were streaked on Luria-Bertani agar containing ampicillin (20 μg/ml) and thymine (50 μg/ml) (a to f), chloramphenicol (25 μg/ml) in addition to ampicillin (20 μg/ml) and thymine (50 μg/ml) (g to j), and ampicillin (50 μg/ml) and thymine (50 μg/ml) (k to m) and were then incubated for 24 h at 42°C.
- FIG. 3.
Deacylation of the arginylated molecules of the wild-type argU tRNA (○ and •) and the argU10(Ts) tRNA (□ and ▪) in the presence (○ and □) or absence (• and ▪) of EF-Tu at 30°C (A) and 43°C (B). Samples were withdrawn after incubation for 0, 10, 20, 30, and 40 min at 30°C and after incubation for 0, 6, 12, 20, and 30 min at 43°C. The relative amounts of arginyl-tRNA are plotted on a log scale against the incubation time.
- FIG. 4.
Arginylation of the wild-type (○) and mutant (•) argU tRNA at 30°C (A) and 43°C (B). Samples were withdrawn after 20, 40, 60, and 90 s of incubation.
- FIG. 5.
argU tRNA arginylation levels in the wild-type cells (A) and mutant cells (B) before and 60 min after the temperature upshift (lanes 1 and 2, respectively). The tRNA extracts analyzed in each lane by acid-urea gel electrophoresis were 2.5- and 5-μg extracts in panels A and B, respectively. The positions of the arginylated and uncharged tRNAs on the gel are indicated by a and b, respectively. The argU tRNA was detected by hybridization to a 32P-labeled specific probe after transfer of tRNAs from the gel onto a nylon membrane. The values below the lanes are the relative levels of tyrosine acceptance for the tRNA extracts at the same concentration.
- FIG. 6.
Interactions of the G1-C72 base pair at the acceptor stem terminus with EF-Tu-GTP (28) (A) and yeast ArgRS (4) (B). EF-Tu and ArgRS are represented by blue and green ribbons, respectively. Nucleotide residues are represented by sticks, and the phosphate-sugar backbone is outlined by pink tubes. The amino acid residues of EF-Tu (A) and ArgRS (B) that interact with residues 1 to 2 and 71 to 72, respectively, are also represented by balls and sticks.
Tables
- TABLE 1.
Growth of the argU10(Ts) mutants and the parental strains at 42°C
Strain Relevant genotype Growth at 42°Ca KN250 supD126(Ts) Growth KN1044 KN250 rnhA-1::Tn3 Growth KN1453 dnaA5(Ts) rnhA-1::Tn3 Growth YT341 dnaA17(Am) supF6(Ts) Leaky growth YT319 dnaA17(Am) rnhA-199(Am) Growth SF1 KN250 purE79::Tn10 argU10(Ts) Leaky growth SF5 KN1044 purE79::Tn10 argU10(Ts) No growth SF7 KN1453 purE79::Tn10 argU10(Ts) No growth SF9 YT341 purE79::Tn10 argU10(Ts) Leaky growth SF131 YT319 purE79::Tn10 argU10(Ts) No growth SF151 YT319 argU10(Ts) No growth ↵ a Colonies grown at 30°C were streaked on Luria-Bertani agar, and growth was examined after incubation for 24 h at 42°C.
