Construction of Escherichia coli amber suppressor tRNA genes

Effects of mutations at position 36 of tRNAGlu on missense and nonsense suppression in Escherichia coli

“…Further experimentation led to the conclusion that the lack of strong suppressor activity was attributable to a defect in codon recognition or tRNA-ribosome interaction, rather than to a defect in aminoacylation [7]. Similar conclusions were reached during analysis of amber suppressor derivatives of tRNA ~u containing a C34/A36 double mutation [8,9].…”

“…The alternative explanation, that the U36 mutant is completely unrecognized by GluRS, is less appealing and is inconsistent with the observed (albeit low level) production of fl-galactosidase activity in CSH106. Previous studies with tRNAG~UA36(SuUAA/G) [8,9], tRNAG1uC34/A36(SuUAG) (amber suppressor) [7], and tRNAC~UG37 [20], demonstrated that at least some alterations in the anticodon permit recognition by GIuRS. Mischarging by LysRS is also unlikely since tRNA Glu contains several negative determinants for lysine identity [20].…”

“…More efficient tRNAQ~U(SuUAA/G) [7] and tRNAG~u(SuUAG) [8,9] derivatives have been constructed in vitro by introduction of mutations in the anticodon loop and stem. Such mutations may be necessary to facilitate efficient tRNA-ribosome interactions or tRNA-tRNA interactions during translation.…”

Trans-acting RNA inhibits tRNA suppressor activity in vivo

“…Secondary structures of tRNA leu amber suppressor (A), aptamer 1 (B), and aptamer 2 (C), represented as a tRNA-like molecule+ Under each aptamer the linear sequences corresponding to the anticodon loop of the suppressor tRNA are aligned with the corresponding sequences of the aptamer+ Complementary sequences are indicated in bold+ tRNA suppressor inhibition 905 complementary to the anticodon loop of the suppressor are flanked by sequences one would expect not to pair; the 5 nt at the 39 end are also complementary to the 59 half of the anticodon stem of the suppressor (Fig+ 1C)+ When the loops of aptamer 2 and of the suppressor interact, the 39 part of the aptamer's stem is free to base pair with the 59 part of the suppressor's stem; the aptamer-suppressor transitory coupling can lead to the formation of a more stable 12-bp interaction (Persson et al+, 1990;Zeiler & Simons, 1998)+ We cloned both aptamer 1 and 2 into pGFIB-I (Normanly et al+, 1986(Normanly et al+, , 1990)+ In this high-copy number plasmid that encodes an ampicillin-resistant gene, each aptamer is constitutively expressed from a strong synthetic promoter based on the constitutive promoter sequence of the E. coli lipoprotein gene lpp+ Distal to the promoter and restriction site polylinker is a transcription terminator, corresponding to the termination sequence of the ribosomal RNA operon rrnC+…”

“…coli strain XAC-1 is F9 lacI 373 Z u118am proB ϩ /⌬(lac-proB) x111 nalA, rif, ara, argE am + Plasmids pGFIB-I and pDA5YQ are as described by Normanly et al+ (1986Normanly et al+ ( , 1990)+ Strains were routinely grown at 37 8C in Luria-Bertani medium (LB; Difco), supplemented with ampicillin (50 mg/mL; Sigma) in the presence of pGFIB-I and with chloramphenicol (30 mg/mL; Sigma) in the presence of pDA5YC+ Lactose MacConkey agar (Difco) was used to isolate XAC-1 revertants of lac Z amber mutants+ 5-bromo-4-chloro-3-indolyl-D-galactoside (X-gal, 40 g/mL) and isopropyl-D-thiogalactoside (IPTG) were added when appropriate+ We also carried out experiments to verify the stability and phenotype characteristics of the F9 episome in XAC-1 cells+ XAC-1 cells harboring F9 lacI 373 Z u118am proB ϩ /⌬(lac-proB) produced white colonies on lactose MacConkey agar because of the amber mutation in the lac Z gene+ With the aim of isolating colonies in which the amber codon was reverted to a sense codon, we plated XAC-1 cells at 10 Ϫ4 and 10 Ϫ5 dilutions on lactose MacConkey agar, and after prolonged incubation, isolated several small revertant red colonies+ We synthesized two primers flanking the amino acid 17 position in lac Z (see below)+ Using PCR, we amplified DNA from both a XAC-1 white colony and from a purified revertant red colony+ The sequence of the PCR product confirmed the reversion of the amber UAG triplet to CAG+ Oligodeoxyribonucleotides and sequencing DNA oligonucleotides were synthesized on an Applied Biosystems DNA/RNA Synthesizer 392 and purified on denaturing polyacrylamide gels+ With reference to the lac Z sequence (AC J01636), the primers used to sequence the amber mutation and the revertant at the position of amino acid 17 of lac Z in F9 of XAC-1 strain were the following: 59-ATGACCATGATTACGGATTC-39 (nt 1283-1303), 59-GCTATTACGCCAGCTGGCGAAAG-39 (nt 1403-1381)+ The start codon of lac Z corresponds to nt 1284-1286+…”