J.D. Smith

Duplicate genes for tyrosine transfer RNA in Escherichia coli

Abstract Genetic and biochemical studies of Escherichia coli and the new transducing phage φ80psuIII+ have been used to characterize the tRNA genes of E. coli. The transducing phage stimulates the production of both suIII+ and suIII− tyrosine tRNA upon infection, and in hybridization experiments its DNA is saturated with 1.4 tyrosine tRNA molecules per genome. One of its derivatives, selected for its genetic properties, stimulates only suIII+ tyrosine tRNA, and its DNA is saturated by 0.6 tyrosine tRNA molecule per genome. We conclude that the original phage carries two tyrosine tRNA genes, one suIII+ and one suIII−, while the derivative carries a single suIII+ gene. The single-gene derivative apparently arises by unequal recombination involving the two genes of the original phage; the reciprocal recombination product, carrying three tyrosine tRNA genes, is also detected. Entirely analogous single-gene and three-gene derivatives of E. coli are found, and we conclude that E. coli normally carries a pair of closely-linked genes specifying its minor, or suIII tyrosine tRNA.

More mutant tyrosine transfer ribonucleic acids

Abstract The isolation and properties of several mutants of the su III tyrosine suppressor transfer RNA gene are described. Two mutants with temperature-sensitive suppressor activity specify tRNAs with single base substitutions, giving mispaired bases in the amino acid acceptor arm. In one of these a G · C pair has been replaced by an A · C pair. A second site revertant of this mutant to wild-type suppressor activity specifies a tRNA with an A · U pair at this position. A similar two-step conversion of a G · C pair in the arm of the dihydrouracil loop to an A · U pair is described. Changing either of these base pairs from G · C to A · U does not alter the K m of aminoacylation of the tRNA with tyrosine tRNA synthetase. The tRNA sequence changes associated with three other second-site revertants are described. These have nucleotide substitutions in the dihydrouracil loop and arm. In two instances a C residue is changed to a dihydrouridylic acid residue. Only small amounts of tRNA are synthesised in vivo from the single-step su III mutants, including those specifying mutant tRNAs with A · C pairs in helical regions. In these, restoration of the normal hydrogen bonding by a second mutation results in restoration of the normal levels of tRNA synthesis. A second mutation which increases the synthesis of tRNA in a temperaturesensitive mutant is described. This mutation is linked to the su III − gene but does not lie within the tRNA sequence.

Mutant tyrosine transfer ribonucleic acids.

Three independent mutants of the suIII tyrosine suppressor transfer RNA gene have been isolated. These mutants are shown to produce mutant tRNAs differing from the wild-type suIII+ tRNA molecule in each case by a single base change. A mutant tRNA containing an A residue in place of a G in the “dihydrouracil loop” appears to be defective in a step in protein synthesis occurring after the acylated tRNA is bound to the ribosome. A mutant tRNA having an A residue in place of a G in the “anticodon stem” appears to be defective exclusively in its apparent affinity for the tyrosyl tRNA synthetase. The isolation of mutant tRNAs as well as their biological properties are discussed.

Specific hybridization of tyrosine transfer ribonucleic acids with DNA from a transducing bacteriophage φ80 carrying the amber suppressor gene suIII

Abstract Transducing phages of φ80 have been isolated which carry the su III gene. We have used the technique of DNA-RNA hybridization to test the hypothesis that a tRNA Tyr molecule is the direct product of this gene. The following results strongly support this hypothesis: 1. (1) Upon infection of Escherichia coli by φ80 d su III , there is a tenfold increase in the fraction of the tRNA which will hybridize with φ80 d su III DNA. This is to be compared with a similar increase in the fraction of tRNA which will accept tyrosine following infection. 2. (2) Saturation experiments show that there is a single nucleotide sequence (a gene) which will hybridize with tRNA. 3. (3) As tRNA is purified for tyrosine-accepting activity, there is an increase in the fraction of the tRNA which will hybridize with φ80 d su III DNA. This has been shown both in saturation and in competition experiments. Our view of suppression dictates that there be at least two genes specifying tyrosine tRNAs in E. coli . One of the tRNAs, the major species, is not involved in suppression; the other, a minor species, recognizes the normal tyrosine codons in the su − cell, and UAG, a chain-terminating codon in the su + III cell. The present evidence shows that these two tRNA molecules must be very similar, as they both hybridize efficiently with φ80 d su III DNA.

Structure of ribonucleic acid.

fundamentals, and showed by adapting the equations of Lagrange that in certain systems the part played by energy in linear systems is played by areas under a curve in the case of non-linear systems. Manual control as distinct from automatic control was not forgotten. The paper on this subject by J. D. North included a thorough analysis of a model that may provide a useful approximation for the response of hand to eye in some simple situations such as target tracking or car driving. The discussions throughout the conference were vigorous and sustained. It appeared to be the opinion of all who attended that the conference has helped to lay foundations on which much further progress may now be based. The papers presented, together with selected material from the discussions during the conference, will be published in due course as a single volume by Butterworths Scientific Publications, Ltd. A. TUSTIN

Action of puromycin in polyadenylic acid-directed polylysine synthesis

The action of puromycin on the polyadenylic acid-directed synthesis of polylysine by the Escherichia coli amino acid incorporation system has been studied. The homologous series of lysine oligopeptides formed are normally attached to sRNA. Addition of puromycin leads to the accumulation of lysine-containing compounds unattached to sRNA. These have been separated by chromatography, digested with trypsin and their products examined. They have the expected properties of lysine oligopeptides attached through their carboxyl group to puromycin by peptide linkage. Further evidence for attachment of lysine peptides to puromycin has been obtained by using an analogue of puromycin, puromycin 5′- β -cyanoethylphosphate. Incubation of 32 P-labelled puromycin 5′- β -cyanoethylphosphate with the system in the presence of 3 H-labelled lysine gave a series of compounds, labelled both with 32 P and 3 H, analogous to those obtained with puromycin. These were separated by chromatography and contained 32 P and 3 H in ratios expected from lysyl peptides terminated by a single puromycin analogue residue. No significant amounts of lysine peptides unattached to puromycin were released from sRNA in the presence of puromycin. The major products were di- and trilysyl puromycin with smaller amounts of higher homologues. No detectable monolysyl puromycin was formed (on incubation of the system in the presence of puromycin and polyadenylic acid).

Transcription and processing of transfer RNA precursors.

Publisher Summary This chapter discusses the transcription of tRNA genes as precursors and how these are converted to mature tRNA. One important step in tRNA biosynthesis, the modification of tRNA bases, is discussed only in relation to the precursors. In the chapter, the term precursor is used to indicate the original transcript or any intermediate in the size reduction of this to tRNA, processing denotes any of the nucleolytic cleavages in these steps, and the term modification is reserved for the various substitutions and rearrangements that yield the modified nucleoside residues in tRNA. The primary transcripts are longer RNAs containing extra sequences not present in the functional molecules. In the case of the mammalian ribosomal RNA precursor, three separate ribosomal RNA components are cotranscribed. The chapter reviews that the tRNA precursors were first found in mammalian cells, but most of the knowledge about these steps in the biosynthesis of tRNA has come from Escherichia coli, where a combination of genetic analysis and nucleotide sequencing not only has revealed much about how the tRNAs are transcribed, but also has led to the discovery of a set of highly specific nucleases involved in the cleavage of the tRNA gene transcripts.

Mischarging in mutant tyrosine transfer RNAs

The suIII gene of E. coli offers a good system for the isolation of mutant tyrosine transfer RNAs [l-4] . Alteration by mutation of the amino-acid acceptor specificity of a tRNA would provide a method of investigating the nature of the site on a tRNA recognised by its cognate aminoacyl-tRNA synthetase. We here describe properties of two previously isolated suIII mutants which strongly suggest that their acceptor specificity is thus altered, and the isolation of two new mutants having similar properties. The sites of all four mutations are tightly clustered in stem (a) of the cloverleaf structure.

Selective reaction of methoxyamine with cytosine bases in tyrosine transfer ribonucleic acid

Abstract The effect of pH on the rate and product ratio has been investigated for the reaction of methoxyamine with cytidine. In the pH range 5.0 to 5.5, approximately 20% of the cytosine bases in mixed Escherichia coli transfer RNA are reactive towards methoxyamine. The cytosine bases in the su + III tyrosine suppressor tRNA which are reactive towards methoxyamine have been characterized as C-16, 19, 35, 51, 83 and 84. Surprisingly, residue C-33, a base at the 5′ terminal of the anticodon loop, is relatively resistant to modification. These results are discussed in terms of transfer RNA conformation.

Identification of an ochre-suppressing anticodon

Abstract An amber suppressing tyrosine transfer RNA has been converted to an ochre suppressor by hydroxylamine mutagenesis. Nucleotide sequence analysis reveals that the anticodon of the new suppressor is UUA as expected.