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By DNA sequencing and gene dissection, it has been revealed that Su+UGA#11, the mutant prfB of E. coli (Chang et al., 1990) has a double mutation compared with the wild-type LS653: one is a base substitution from T to C at the codon 63 and the other is from G to A at the codon 79. Both mutations cause amino acid substitution, Leu63----Phe63 (L63F) and Asp79----Gly79 (D79G), and are necessary to confer the efficient UGA suppressor activity.

UGA-specific nonsense suppressors from Escherichia coli K-12 were isolated and characterized. One of them (Su+UGA-11) was identified as a mutant of the prfB gene for the peptide releasing factor RF2. It appears that in this strain, while peptide release at sites of UGA mutations is retarded, the UGA stop codon is read through even in the absence of a tRNA suppressor, exhibiting a novel type of passive nonsense suppression. Three suppressors (Su+UGA-12, -16 and -34) were capable of restoring the streptomycin sensitive phenotype in resistant bacteria (strAr). Because of their drug-related phenotype, these are possibly mutations in the components of the ribosomal machinery, particularly those concerned with peptide release at UGA nonsense codons. A tRNA suppressor was also obtained which was derived from the tRNA(Trp) gene. In this strain, a long region between rrnC (84.5 min) and rrnB (89.5 min) was duplicated and one of the duplicated genes of tRNA(Trp) was mutated to the suppressor. The mechanism of UGA-suppression is discussed in terms of translation termination at the nonsense codon in both active and passive fashions.

DNA molecules of phi 80sus2psu3+ and phi 80dsu3+ isolated by Andoh and Ozeki (1968) were studied by the electron microscope heteroduplex method. The phi 80sus2psu3+ and phi 80dsu3+ DNA lengths were found to be 108.7 and 103.3% of the phi 80 DNA, respectively. The phi 80sus2psu3+/phi 80 heteroduplex shows an insertion loop of 8.7% of the phi 80 DNA which migrates from 7.7 to 9.7%, as measured relative to the left (0%) and right (100%) termini of the mature phi 80 DNA molecule. The region of loop migration occupies the central region of the phi 80 head gene cluster. The presence of su3+-containing Escherichia coli DNA of 6.7% phi 80 unit flanked by two homologous regions of phage DNA of 2.0% of phi 80 unit gives rise to a movable insertion loop. In phi 80dsu3+, from which phi 80sus2psu3+ was derived, 50.5% of the phi 80 DNA at the left arm was replaced by E. coli DNA containing the su3+ gene, equivalent to about 53.8% phi 80 unit in length. The phi 80sus2psu3+/phi 80dsu3+ heteroduplex appears as a double-stranded molecule that bifurcates into two clearly visible single-stranded regions, rejoins, bifurcates, and rejoins again. The middle double-stranded stretches of 6.7% phi 80 unit correspond to the E. coli DNA inserted in phi 80sus2psu3+. Therefore the transducing fragment carried by phi 80sus2psu3+ originates from the inside region of the transducing fragment of defective phage phi 80dsu3+ by at least two illegitimate recombination events.

An Escherichia coli DNA fragment containing an Su+6 amber suppressor gene (supP) was cloned into a lambda gt lambda Ch vector by the shotgun method, selecting a Su+6 transducing phage lambda pSu+6. Through prophage integration followed by induction occurring at the transducing region of the lambda pSu+6 in Su- E. coli, a counterpart transducing phage carrying the wild-type allele (Su degrees 6) was isolated (lambda pSu degrees 6). The fingerprint of a tRNA encoded by lambda pSu degrees 6 was identical to that of an unidentified tRNAE previously reported (Ikemura & Ozeki, 1977). The cloverleaf structure of this tRNA was determined by combining the results of tRNA analysis and DNA sequencing of the gene. Judging from the anticodon of 5'-CAA-3', Su degrees 6 tRNA was identified as a new type of leucine isoacceptor in E. coli. Unlike other suppressors analyzed, Su+6 tRNA differed by two nucleotides from Su degrees 6 tRNA; one at the anticodon (CAA to CUA) and the other at the junction of D- and anticodon-stem (A27 to G27). DNA sequence analysis revealed that a single stretch of tRNA is flanked by the putative sequences of promoter and terminator. Thus a single copy of the Su degrees 6 tRNA gene constitutes a solitary tRNA transcription unit. Southern blotting showed only one copy of Su degrees 6 tRNA gene per haploid genome of E. coli. Since this single gene can mutate to the Su+6 suppressor, the Su degrees 6 leucine tRNA may be accounted as a dispensable species among the leucine isoacceptor tRNAs. Two possible open reading frames are found immediately following the Su degrees 6 tRNA gene.

Transducing phages of lambda carrying suppressors, lysT (Su+ beta), supG and and supL, were isolated in vivo. Upon infection with each of these phages, the production of tRNALys and tRNAVal1 was markedly enhanced. Fingerprint analysis of these tRNAs revealed that they consisted of normal tRNALys, mutant tRNALys and tRNAVal1 in equimolar ratios. The mutant tRNALys carried a single-base alteration at the anticodon, from 5'-UUU-3' to 5'-UUA-3', which makes it an ochre suppressor. DNA sequence analysis of the entire transducing fragment (730 base-pairs) of lambda pSu+ beta revealed that three tRNA genes are tightly clustered within a transcription unit in the following order; i.e. promoter-(48 base-pairs)-wild-type tRNALys-(132 base-pairs)-tRNAVal1-(2 base-pairs)-Su+ beta tRNALys-. In wild-type bacteria there are two identical tRNALys genes in one operon. Although we have shown that in Su+ beta it is the distal tRNALys that has been mutated to the ochre suppressor by a single base change at the anticodon (U36 to A36), we have not determined which of the two genes bears the supG or the supL mutation. The sequences following both tRNALys genes are highly homologous: both are about 100 base-pairs long and both terminate with an 18 base-pair sequence homologous to the last 18 bases of each tRNA. The sequences of tRNALys and tRNAVal1 are also very similar. Thus, including the 3'-portions of these tRNA genes, the 18 base-pair sequence is more or less periodically repeated five times in the DNA sequence.