UGA Suppression Research Articles

Regulation of the [formula omitted] tryptophan operon by readthrough of UGA termination codons

The regulation of the synthesis of trp operon enzymes was studied in streptomycin-resistant Escherichia coli mutants temperature-sensitive for UGA suppression by normal tRNA Trp. Our mutants carry a trp R + allele that when transferred to a different genetic background causes repression of trp operon enzyme synthesis at both low (35°C) and high (42°C) temperatures; however, in our mutants with an excess of tryptophan and at increased temperatures trp enzyme synthesis is derepressed. Based on our results and the sequence data of the trp R gene [Singleton et al. (1980) Nucleic Acids Res., 8, 1551–1560], we offer a model for the involvement of the limited misreading of UGA codons by normal charged tRNA Trp in the autogenous regulation of the trp R gene expression. The UGA readthrough process may be a regulatory amplifier of the effect of tryptophan starvation.

UGA suppression by normal tRNA Trp in Escherichia coli: codon context effects.

The nucleotide sequences at the 3' side of in-phase UGA termination codons in mRNAs of various prokaryotic genes were re-examined. An adenine (A) residue is found to be adjacent to the 3' side of UGA in mRNAs which code for readthrough proteins by the suppression of UGA by normal Escherichia coli tRNA Trp. It is suggested that the nature of the nucleotide following a UGA codon determines whether the UGA signals inefficiently or efficiently the termination of polypeptide chain synthesis: an A residue at this position permits the UGA readthrough process.

A UGA termination suppression tRNATrp active in rabbit reticulocytes.

A tRNATrp was purified from rabbit reticulocytes which suppresses the UGA termination codon of beta-haemoglobin mRNA. Evidence is presented that the beta-haemoglobin readthrough protein is found in reticulocyte translations and intact cells. Some natural readthrough proteins perform essential functions; they are synthesised through suppression of UGA or UAG but not UAA termination codons.

Effect of photochemical crosslink S 4U(8)-C(13) on suppressor activity of su + tRNA Trp from Escherichia coli

Readthrough in vitro of the Qβ coat protein terminator codon UGA has been used as an assay for suppression by UGA-suppressor tRNA Trp. When the tRNA is covalently crosslinked between 4-thiouracil(8) and cytosine(13) by irradiation at 334 nm, it is found that UGA suppression by this assay is reduced to the low level characteristic of the wild type tRNA Trp. In contrast, crosslinking has little effect on incorporation of tryptophan in response to UGG codons. Thus, incorporation of tryptophan during translation of R17 messenger RNA is unaffected by photochemical crosslinking. Furthermore, dilution experiments using R17 mRNA in which tryptophan incorporation is dependent on precharged suppressor Trp-tRNA show that the crosslinked species competes well with non-irradiated tRNA. These results further emphasize the influence on tRNA-ribosome interactions of the region in tRNA around the dihydrouridine arm, where the mutation, in the suppressor is found and the photochemical crosslink is introduced.

Anticodon conformation and accessibility in wild-type and suppressor tryptophan tRNA from E. coli.

The association between Trp-tRNA and Pro-tRNA, which have complementary anticodon sequences, has been used as a probe of anticodon conformation. It is unaffected, however, by the base change in the D-stem present in UGA-suppressor Trp-tRNA. This does not support the hypothesis that UGA suppression depends upon a conformational change induced in the anticodon. The stable denatured form of wild-type Trp-tRNA no longer interacts with Pro-tRNA; the structure of the anticodon region must therefore be quite different in the denatured form.

UGA and non-triplet suppressor reading of the genetic code.

Mutants in a tRNA methylase gene, supK, of Salmonella typhimurium give suppression of UGA and certain frameshift mutants. In addition suppressors which do not suppress UGA, but which suppress frameshift mutants of opposite (effective) signs are described.

Tryptophan transfer RNA as the UGA suppressor

Genetic data indicate that tryptophan is inserted during suppression of the UGA nonsense codon by the Escherichia coli strain, CAJ64. The nucleotide sequences of tryptophan transfer RNA's from CAJ64 and the related wild-type strain, CA244, are identical except at position 24 in the stem of the dihydrouracil loop. CA244 tRNA Trp has G at this position; CAJ64 tRNA Trp has A. The anticodons are CCA, expected for recognition of the tryptophan codon, UGG, alone. Tryptophan tRNA from several related su − strains denatures upon isolation, whereas that from suppressor strains is stable. An independently isolated UGA suppressor strain has tRNA Trp identical to CAJ64. It is concluded that UGA suppression is carried out by a tRNA Trp whose anticodon-codon recognition is altered by a change elsewhere in the molecule.