Ingvar Svensson

STUDIES ON MICROBIAL RNA. I. TRANSFER OF METHYL GROUPS FROM METHIONINE TO SOLUBLE RNA FROM ESCHERICHIA COLI.

Two partially purified enzyme preparations, one from yeast and one from Escherichia coli, have been used to study the transfer of methyl groups from methionine to soluble RNA (S-RNA) from E. coli. The reaction requires ATP and magnesium. The E. coli enzyme can incorporate methyl groups only into S-RNA from methionine-starved E. coli K12 W6 (a mutant with a deficient control of RNA synthesis). The yeast enzyme can methylate S-RNA from E. coli K12 in log phase as well as from methionine-starved cultures of W6. The methylation is not inhibited by periodate oxidation of the terminal adenosine residue in the S-RNA. The alkaline stability of the methylated S-RNA is clearly different from that of methionyl RNA. Methylated S-RNA has been degraded by snake venom enzymes as well as by alkaline hydrolysis. The digestion products have been examined by paper and anion exchange chromatography, respectively. In vitro, neither of the two enzymes can methylate purified ribosomal RNA (R-RNA). In vivo incorporation of methionine has, however, shown a significant methylation of both S-RNA and R-RNA. The composition of the methylated nucleosides obtained from R-RNA differs from that of S-RNA. Total RNA contains also smaller amounts of non-methylated RNA. Counter-current distribution of S-RNA has shown that the acceptors for the methyl groups can be separated from the acceptors for tyrosine.

Aminoacylation and polypeptide synthesis with tRNA lacking ribothymidine

Abstract The role of thymine in tRNA has been investigated in vitro , using a mutant of Escherichia coli completely lacking thymine in its tRNA. In both aminoacylation and polypeptide synthesis, the thymine-free tRNA seems to behave identically with normal tRNA.

Studies on methionyl-tRNA synthetase I. Effects of divalent and monovalent cations on methionyl-tRNA synthetase from Saccharomyces cerevisiae

Abstract 1. 1.|The activation of methionyl-tRNA synthetase ( l -methionine:tRNA ligase (AMP), EC 6.1.1.10) from Saccharomyces cerevisiae by divalent metal ions has been investigated. It has been shown that the concentration for optimal reaction rate varies with the kind of cation and also with the kind of tRNA used as substrate. 2. 2.|In the presence of NH4+, K+ or Rb+ the rate of methionyl-tRNA formation is drastically increased, while the rate of methionyl-AMP formation is almost unaffected. 3. 3.|Some models for the mechanism of monovalent cation action are briefly discussed.

Studies on methionyl-tRNA synthetase III. Enzyme dependence for maximum methionyl-tRNA formation

Abstract 1. 1. In the heterologous reaction between methionyl-tRNA synthetase ( l -methionine:tRNA ligase (AMP), EC 6.1.1.10) from Saccharomyces cerevisiae and tRNA from Escherichia coli , the final yield of methionyl-tRNA varies with the enzyme concentration. 2. 2. The yield is also influenced by the presence of monovalent cations.

Two transfer RNA (1-methylguanine) methylases from yeast

Two distinct tRNA (m-1G) methylases have been found in the yeast Saccharomyces cerevisiae. They differ in their chromatographic properties on hydroxyapatite, in their response to spermine, and in their site specificity. Only one of the methylases is active against normal tRNA from Escherichia coli.

Studies on methionyl-tRNA synthetase II. Effects of divalent and monovalent cations on methionyl-tRNA synthetase from Escherichia coli

Abstract 1. 1.|The influence of divalent and monovalent cations on the activity of methionyl-tRNA synthetase ( l -methionine:tRNA ligase (AMP), EC 6.1.1.10) from Escherichia coli has been studied, and a comparison has been made with the corresponding enzyme from yeast. 2. 2.|Of the monovalent cations only NH4+ stimulates the rate of methionyl-tRNA formation. This effect is found with both homologous and heterologous tRNA. The magnitude of the stimulation varies with pH. 3. 3.|The results explain a phenomenon previously interpreted in terms of a “regenerating” enzyme in amino acid activation.

Chromatographic fractionation of Escherichia coli transfer RNA of a new support, naphthoyl-Sepharose

The noncharged naphthoyl-Sepharose CL-6B has been prepared. Escherichia coli tRNA binds to this new adsorbent in 0.75 M ammonium sulphate at neutral pH at room temperature. Using a negative salt gradient, the tRNAs are eluted in a defined order. The chromatographic pattern is clearly different from those of other commonly used tRNA separation techniques.

Hydrophobic interaction chromatography of incompletely methylated transfer RNA from escherichia coli on octyl-sepharose

Phenylalanine-specific transfer RNA from methionine-starved relaxed Escherichia coli K12 separates into two components when chromatographed on Octyl-Sepharose. The difference in elution between the two tRNAs has been shown to depend on the methyl group in the highly modified 2-methylthio-N-6-isopentenyladenosine. The first eluted tRNAPhe lacks this methyl group, while the last eluted tRNAPhe is fully methylated. Other differences in the modification patterns have no effect on the elution from Octyl-Sepharose. The elution pattern of tyrosine- and serine-specific tRNAs, also normally containing ms2i6A, is similar.

Site specificities of three transfer RNA methyltransferases from yeast

The site specificities of two distinct tRNA(m1G)methyltransferases and one tRNA(m2G)methyltransferase from yeast have been investigated by heterologous methylation and analysis of purified Escherichia coli tRNAs. The two tRNA(m1G)methyltransferases were found to be specific for sites 9 and 37, respectively. The tRNA(m2G)methyltransferase was specific for site 10. Two of the enzymes were purified by affinity chromatography on tRNA-Sepharose.

Artificial zoning in anion exchange chromatography

Abstract 1. A study has been made of step-wise and gradient development in anion exchange chromatography. 2. In one-step development the anomalous step previously studied has been shown to arise from the anions adsorbed on the column during equilibration. 3. The deformation of concentration gradients by passage through the column has been demonstrated. 4. By the use of a combined pH and concentration gradient with mixed buffers highly purifid bovine carbonic anhydrase has been split up into two active peaks, which are probably identical. 5. The possibilities of obtaining artificial protein peaks in one-step and gradient development chromatography on anion exchangers are discussed.