M.P. Stulberg

Isolation and properties of undermethylated phenylalanine transfer ribonucleic acids from a relaxed mutant of Escherichia coli

Abstract The function of methylated bases in transfer RNA (tRNA) was investigated with purified components of the phenylalanine-activating system. Reversed phase column chromatography was employed for the separation of tRNA Phe from mixtures of tRNAs synthesized by a relaxed mutant of Escherichia coli (met − ) before and after methionine starvation. Normal and undermethylated tRNA Phe were isolated and were characterized by their degree of methylation, amino acid-accepting capacity, and by end-group analysis. Kinetic data on initial velocities at saturating concentrations of tRNA indicated that the undermethylated species were considerably less reactive than the normal tRNA Phe .

Restoration of aminoacylation activity of undermethylated transfer RNA by in vitro methylation

Abstract Undermethylated tRNA isolated from a RCrel mutant of Escherichia coli has been examined to determine whether its capacity to accept amino acids was comparable to that of normal tRNA from the same organism. Previously, little, if any differences have been reported (see Littauer and Milbauer, 1965 , Borek and Srinivasan, 1966 ). In this communication we report the restoration of aminoacylation activity of undermethylated tRNA by in vitro methylation. In a mixture of undermethylated tRNAs 2 from E. coli , we found that for four amino acids tested (Phe, Leu, Tyr, and His), the corresponding tRNAs showed decreased aminoacylation activities when compared to normal tRNA. Upon in vitro methylation of the undermethylated tRNAs with methylases free of RNase, an increase in the aminoacylation activities occurred. With two tRNA species (Phe and His), complete restoration of aminoacylation activity was observed.

[2] Reversed-phase chromatography systems for transfer ribonucleic acids — preparatory-scale methods☆

Publisher Summary This chapter describes the reversed-phase chromatography (RPC) systems; various types of RPC systems; the general effect of experimental variables, such as pH, temperature, load, and magnesium ion concentration; and describes several specific examples of the application of RPC columns to both small- and large-scale separation and recovery of purified tRNAs. The RPC-2 system has the most even distribution of tRNAs throughout the chromatogram from the front to the rear, which makes this system particularly useful in studying the heterogeneity of tRNAs. Another advantage is the relatively long life of a column because of the very low aqueous solubility of the fluorocarbon diluent and quaternary ammonium chloride. The RPC-3 system has proved to be particularly useful for separation of tRNAs near the front of the chromatogram; tRNAs can be readily resolved with this system, which do not separate in any other systems. The RPC-4 system is most useful for the separation of tRNAs that run near the rear of the chromatogram, but tends to give less separation of tRNAs near the front of the chromatogram. The RPC systems are used for the separation, and purification of a variety of tRNAs from different organisms.

Modified nucleosides in undermethylated phenylalanine transfer RNA from Escherichia coli.

A complete nucleoside analysis of highly purified undermethylated and normal tRNAPhe from a methionine-starved culture of Escherichia coli RCrel showed that the undermethylated species contained N6-isopentenyladenosine in place of 2-methyl-thio-N6-isopentenyladenosine. This difference may account for the inactivity of undermethylated tRNAs in protein synthesis. Both normal and undermethylated tRNAPhe contained fractional amounts of 7-methylguanosine. The amount of unmodified uridine in undermethylated tRNAPhe was in excess of that expected due to lack of methylation.

Modification of 4-thiouridine and phenylalanine transfer RNA with parachlormercuribenzoate☆

Abstract While investigating the modification of the 4-thiouridine moiety in E. coli tRNAPhe with parachloromercuribenzoate with a view to further elucidate the biochemical function of the minor base and prepare tRNA labeled with a heavy metal at a specific site for X-ray crystallography, we observed that the paramercuribenzoate moiety in the modified tRNAPhe is very labile in the presence of Mg2+. Surprisingly this lability is not observed at the mononucleoside level when the reaction is carried out with the mercurated derivatives of 4-thiouridine or its methyl analog. Evidences for 1:1 stoichiometry of the reaction of parachloromercuribenzoate with 4-thiouridine and for the covalent nature of the HgS bond in the mercurated nucleoside have been obtained by carrying out the reaction with N1-methyl-4-thiouracil and isolating and characterizing the product, 1-methyl-4-thiouracilylparamercuribenzoic acid.

[39] Isolation, purification, and methylation of undermethylated tRNAPhe from an RCrel mutant of Escherichia coli☆

Publisher Summary This chapter describes the isolation, purification, and methylation of undermethylated transfer ribonucleic acid (tRNA) Phe from an RC rel mutant of Escherichia coli . Undermethylated tRNA is obtained from a mutant (RC rel ) of the bacterium Escherichia coli that exhibits the phenomenon of “relaxed” control over RNA synthesis. Under appropriate culture conditions, the mutant can be made to synthesize tRNA that is free of methylated bases; however, the tRNA extracted from the organism under these culture conditions is a mixture of normal, fully methylated tRNA, and undermethylated tRNA. As tRNA obtained from the RC rel mutant represents a mixture of 100 or more tRNA species that are similar in structure, molecular weight, and net charge, the successful separation and purification of an individual undermethylated tRNA species require techniques that can be used to detect subtle differences between similar macromolecules.

An analysis in vivo of histidine transfer RNA during repression and derepression in Bacillus subtilis

Abstract Bacillus subtilis 30 contains two iso-accepting species of tRNAHis that are relatively labile to hydrolysis when aminoacylated. The two species, when studied under repressive and derepressive states of the histidine biosynthetic enzymes, are qualitatively and quantitatively equivalent. However, derepressed cells have an average of 66 % of the histidyl-tRNA present in repressed cells in vivo. This difference is not due to the total amount of tRNAHis present in the two states but may reflect differences in the aminoacylation reaction in vivo.

Undermethylated transfer RNA does not support phage RNA-directed in vitro protein synthesis

A cell-free protein synthesis system from Escherichia coli Q13 was depleted of mRNA and tRNA so that restoration of maximum activity was dependent of the addition of these components. Protein synthesis, directed by either MS2 or Qbeta phage RNA, was stimulated significantly by the addition of normal tRNA from either E. coli Q13 or B. In contrast, undermethylated tRNA from methionine-starved E. coli RCrel did not cause this stimulation. It is concluded that undermethylated tRNA Lacks sufficient base modifications to function in protein synthesis.

Studies on the function of the non-primer tRNAs associated with the 70 S RNA of avian myeloblastosis virus

Significant amounts of three tRNAs are associated with the 70 S RNA of avian myeloblastosis virus (AMV). The temperatures at which they are half dissociated from the 70 S RNA in 50 mM NaCl and their respective quantities relative to 35 S RNA are: tRNAArg, 51 degree C, 1.6; tRNALys, 57 degree C, 0.7 and tRNATrp, 76 degree C, 1.0. Possible functions for the non-primer tRNAs (tRNAArg and tRNALys) were evaluated by determining the effect of their thermal dissociation on: (a) conversion of 70 S to 35 S RNA, (b) capacity of 70 S and/or 35 S RNA to be translated in vitro, and (c) capacity of 70 S and/or 35 S RNA to be reverse transcribed in vitro. Conversion of 70 S to 35 S RNA occurred with a tm of 56 degree C and is consistent with the hypothesis that tRNALys might be involved in joining two 35 S RNA subunits to form the 70 S RNA complex. There was no indication that the association of either tRNAArg or tRNALys influenced the rate or quality of translation of 70 S or 35 S RNA. A decrease in the rate at which 70 S RNA is transcribed occurs in parallel with the dissociation of tRNAArg and tRNALys.

Differential effects on RNA translation by a KCl extract of reticulocyte ribosomes: Characteristics of an inhibitory fraction

Abstract A KCl extract of rabbit reticulocyte ribosomes has been demonstrated to markedly stimulate the translation of various messenger RNAs in a cell-free system from Krebs II ascites tumor cells. In contrast, the translation of encephalomyocarditis viral RNA is strongly inhibited by the same extract. Fractionation of the KCl extract allows the separation of these inhibitory and stimulatory activities. The inhibitory activity has been shown to be the consequence of an unusual endonuclease, associated with ribosomes, that produces approximately 4 S products from the degradation of globin mRNA and viral RNA.