Transgenic Arabidopsis lines (ecotype Col‐0) carrying the Enterobacter agglomerans IaaspH gene under CaMV 35S promoter control were more sensitive to exogenous indole‐3‐acetyl aspartic acid (IAA‐Asp) ...and metabolized 2′‐14CIAA‐Asp more rapidly than control lines. Free IAA, total IAA and IAN levels in independent transgenic lines that accumulated IaaspH mRNA varied insignificantly from control levels, yet IAA‐Asp levels were significantly reduced. The transgenic lines were grown in a variety of conditions and subjected to morphometric analysis. All three lines showed statistically significant differences in rosette diameter (in soil), root and hypocotyl length (on agar). These effects were transient in some cases and did not manifest themselves under all growth conditions tried. The two independent lines with single T‐DNA insertions had lower seed set compared to control lines.
Using synthetic oligonucleotides, we have constructed 17 tRNA suppressor genes from Escherichia coli representing 13 species of tRNA. We have measured the levels of in vivo suppression resulting from ...introducing each tRNA gene into E. coli via a plasmid vector. The suppressors function at varying efficiencies. Some synthetic suppressors fail to yield detectable levels of suppression, whereas others insert amino acids with greater than 70% efficiency. Results reported in the accompanying paper demonstrate that some of these suppressors insert the original cognate amino acid, whereas others do not. We have altered some of the synthetic tRNA genes in order to improve the suppressor efficiency of the resulting tRNAs. Both tRNA(CUAHis) and tRNA(CUAGlu) were altered by single base changes, which generated -A-A- following the anticodon, resulting in a markedly improved efficiency of suppression. The tRNA(CUAPro) was inactive, but a hybrid suppressor tRNA consisting of the tRNA(CUAPhe) anticodon stem and loop together with the remainder of the tRNA(Pro) proved highly efficient at suppressing nonsense codons. Protein chemistry results reported in the accompanying paper show that the altered tRNA(CUAHis) and the hybrid tRNA(CUAPro) insert only histidine and proline, respectively, whereas the altered tRNA(CUAGlu) inserts principally glutamic acid but some glutamine. Also, a strain deficient in release factor I was employed to increase the efficiency of weak nonsense suppressors.
Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress ...an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid. The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.
A leucine transfer RNA has been transformed into a serine transfer RNA by changing 12 nucleotides. This result indicates that a limited set of residues determine tRNA identity.
Amber suppressor genes corresponding to Escherichia coli tRNAPhe and tRNACys have been constructed for use in amino acid substitution studies as well as protein engineering. The genes for either ...tRNAPheGAA or tRNACysGCA both with the anticodon 5' CTA 3' were assembled from four to six oligonucleotides, which were annealed and ligated into a vector. The suppressor genes are expressed constitutively from a synthetic promoter, derived from the promoter sequence of the E. coli lipoprotein gene. The tRNAPhe suppressor (tRNAPheCUA) is 54-100% efficient in vivo, while the tRNACys suppressor (tRNACysCUA) is 17-50% efficient. To verify that the suppressors insert the predicted amino acids, both genes were used to suppress an amber mutation in a protein coding sequence. NH2-terminal sequence analysis of the resultant proteins revealed that tRNAPheCUA and tRNACysCUA insert phenylalanine and cysteine, respectively. To demonstrate the potential of these suppressors, tRNAPheCUA and tRNACysCUA have been used to effect amino acid substitutions at specific sites in the E. coli lac repressor.
Amber suppressor genes corresponding to Escherichia coli tRNA super(Phe) and tRNA super(Cys) have been constructed for use in amino acid substitution studies as well as protein engineering. The genes ...for tRNA super(P) sub(G) super(h) sub(A) super(e) sub(A) or tRNA super(C) sub(G) super(y) sub(C) super(s) sub(A) both with the anticodon 5' CTA 3' were assembled from four to six oligonucleotides, which were annealed and ligated into a vector. The suppressor genes are expressed constitutively from a synthetic promoter, derived from the promoter sequence of the E. coli) lipoprotein gene. The tRNA super(Phe) suppressor (tRNA super(P) sub(C) super(h) sub(U) super(e) sub(A)) is 54-100% efficient in vivo, while the tRNA super(Cys) suppressor (tRNA super(C) sub(C) super(y) sub(U) super(s) sub(A)) is 17-50% efficient. Both genes were used to suppress an amber mutation in a protein coding sequence. NH sub(2)-terminal sequence analysis of the resultant proteins revealed that tRNA super(P) sub(C) super(h) sub(U) super(e) sub(A) and tRNA super(C) sub(C) super(y) sub(U) super(s) sub(A) insert phenylalanine and cysteine, respectively.
We have constructed synthetic genes encoding different Escherichia coli suppressor tRNAs for use in amino acid substitution studies and protein engineering. We used oligonucleotides to assemble the ...genes for different tRNAs with the anticodon 5' CTA 3'. The suppressor genes are expressed from a synthetic promoter derived from the promoter sequence of the E. coli lipoprotein gene. The genes have been used to suppress an amber mutation in a protein coding sequence, and the resulting altered protein has been subjected to sequence analysis to determine the nature of the amino acid inserted at the amber site. Twelve amino acids can now be added in response to the amber codon. We have employed these suppressors to study amino acid substitutions in the lac repressor.
Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as ...determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.
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