Valyl-Transfer RNA: Role in Repression of the Isoleucine-Valine Enzymes in Escherichia coli

alpha-Aminobutyric acid is activated but not transferred to valine-specific transfer RNA and is unable to repress the isoleucine-valine enzymes in Escherichia coli strain W. alpha-Amino-beta-chlorobutyric acid is activated and transferred to valine-specific transfer RNA and completely replaces valine in repression.     

Sulfur: incorporation into the transfer fraction of soluble ribonucleic acid

When S(35)-labeled soluble RNA from Escherichia coli K 38 is subjected to gel filtration, four fractions of RNA are obtained by elution. Only one RNA fraction, the transfer RNA, contains sulfur, presumably as thionucleotides. Treatment with ribonuclease suggests that the incorporated sulfur is an integral part of the polynucleotide chain; digestion with alkali yields a mixture of products containing sulfur, the major one being eluted in a position similar to uridine diphosphate upon Dowex-l chromatography. Analysis by countercurrent distribution of S(35)-labeledtransfer RNA from E. coli B reveals that the incorporated sulfur is found in many RNA's that accept amino acids, but the possibility remains that not all acceptor RNA's contain sulfur.     

Secondary structure of ribosomal RNA

Infrared spectra were obtained for 16S and for 23S ribosomal RNA's in D(2)O solutions. The percentage of each base in the paired and unpaired regions of the RNA was determined from the spectra. The secondary structures of 16S and 23S ribosomal RNA's (from Escherichia coli) are significantly different from each other and are also different from those of yeast ribosomal RNA, formylmethionyl-transfer RNA, and the anticodon fragment of this transfer RNA.     

Cytokinin activity: localization in transfer RNA preparations

Transfer RNA from yeast, liver, and Escherichia coli has cytokinin activity in the tobacco callus bioassay, whereas ribosomal RNA from yeast is inactive. In contrast to fractions of yeast transfer RNA rich in serine acceptor and cytokinin activity, preparations (70 to 90 percent pure) of arginine transfer RNA(2), glycine transfer RNA, phenylalanine transfer RNA, and valine transfer RNA(1) and of highly purified alanine transfer RNA from yeast were inactive at concentrations of 20 to 2500 micrograms per liter. One molecule of 6-(gamma,gamma-dimethylallylamino) purine per 20 molecules of yeast tRNA would account for the observed cytokinin activity. The number of major molecular species contributing to cytokinin activity of transfer RNA, therefore, must be small.     

N-formylseryl-transfer RNA

The reactions between serine and transfer RNA from baker's yeast and from Escherichia coli have been investigated. Results obtained from in vitro, in vivo, and chemical syntheses and from electrophoretic, chromatographic, and radioautographic analyses demonstrate that N-formylseryl-transfer RNA is formed in these systems.     

Specific interactions in RNA enzyme-substrate complexes

Analysis of crosslinked complexes of M1 RNA, the catalytic RNA subunit of ribonuclease P from Escherichia coli, and transfer RNA precursor substrates has led to the identification of regions in the enzyme and in the substrate that are in close physical proximity to each other. The nucleotide in M1 RNA, residue C92, which participates in a crosslink with the substrate was deleted and the resulting mutant M1 RNA was shown to cleave substrates lacking the 3' terminal CCAUCA sequence at sites several nucleotides away from the normal site of cleavage. The presence or absence of the 3' terminal CCAUCA sequence in transfer RNA precursor substrates markedly affects the way in which these substrates interact with the catalytic RNA in the enzyme-substrate complex. The contacts between wild-type M1 RNA and its substrate are in a region that resembles part of the transfer RNA "E" (exit) site in 23S ribosomal RNA. These data demonstrate that in RNA's with very different cellular functions, there are domains with similar structural and functional properties and that there is a nucleotide in M1 RNA that affects the site of cleavage by the enzyme.     

Changing the identity of a tRNA by introducing a G-U wobble pair near the 3' acceptor end

Although the genetic code for protein was established in the 1960's, the basis for amino acid identity of transfer RNA (tRNA) has remained unknown. To investigate the identity of a tRNA, the nucleotides at three computer-identified positions in tRNAPhe (phenylalanine tRNA) were replaced with the corresponding nucleotides from tRNAAla (alanine tRNA). The identity of the resulting tRNA, when examined as an amber suppressor in Escherichia coli, was that of tRNAAla.     

Expanding the genetic code of Escherichia coli

A unique transfer RNA (tRNA)/aminoacyl-tRNA synthetase pair has been generated that expands the number of genetically encoded amino acids in Escherichia coli. When introduced into E. coli, this pair leads to the in vivo incorporation of the synthetic amino acid O-methyl-l-tyrosine into protein in response to an amber nonsense codon. The fidelity of translation is greater than 99%, as determined by analysis of dihydrofolate reductase containing the unnatural amino acid. This approach should provide a general method for increasing the genetic repertoire of living cells to include a variety of amino acids with novel structural, chemical, and physical properties not found in the common 20 amino acids.     

Model substrates for an RNA enzyme

M1 RNA, the catalytic RNA subunit of Escherichia coli ribonuclease P, can cleave novel transfer RNA (tRNA) precursors that lack specific domains of the normal tRNA sequence. The smallest tRNA precursor that was cleaved efficiently retained only the domain of the amino acid acceptor stem and the T stem and loop. The importance of the 3' terminal CCA nucleotide residues in the processing of both novel and normal tRNA precursors implies that the same enzymatic function of M1 RNA is involved.     

Changing the acceptor identity of a transfer RNA by altering nucleotides in a "variable pocket"

The specificity of tRNA(Arg) (arginine transfer RNA) for aminoacylation (its acceptor identity) were first identified by computer analysis and then examined with amber suppressor tRNAs in Escherichia coli. On replacing two nucleotides in tRNA(Phe) (phenylalanine transfer RNA) with the corresponding nucleotides from tRNA(Arg), the acceptor identity of the resulting tRNA was changed to that of tRNA(Arg). The nucleotides used in the identity transformation occupy a "variable pocket" structure on the surface of the tRNA molecule where two single-stranded loop segments interact. The middle nucleotide in the anticodon also probably contributes to the interaction, since an amber suppressor of tRNA(Arg) had an acceptor identity for lysine as well as arginine.     

Construction and recovery of viable retroviral genomes carrying a bacterial suppressor transfer RNA gene

The integration of retroviral genomes into cellular DNA can induce mutations by altering the expression of nearby cellular genes and can serve to identify the gene affected. The construction of a retrovirus that stably carries a suppressor transfer RNA gene from Escherichia coli has allowed facile recovery of the viral genome in vectors marked with amber mutations. This virus can be used for rapid isolation of cellular sequences at the site of proviral insertion.     

Thiobases in Escherchia coli Transfer RNA: 2-Thiocytosine and 5-Methylaminomethyl-2-thiouracil

Two new sulfur-containing pyrimidine nucleotides have been isolated from hydrolyzates of Escherichia coli transfer RNA. The structures, 2-thiocytosine and 5-methylaminomethyl-2-thiouracil, have been assigned to the bases as a result of study of ultraviolet and mass spectra. An acid-degradation product, S-methylamino-methyluracil, has been synthesized and is identical to that derived from the natural product.     

Nucleoside triphosphate termini from RNA synthesized in vivo by Escherichia coli

Alkaline hydrolyzates of RNA made in vivo by Escherichia coli contain ribonucleoside-3'-monophosphate-5'-triphosphates. These probably arise by hydrolysis of the initial nucleoside triphosphate from the 5' terminus of the nascent RNA chains. Logarithmically growing cultures, labeled for 45 seconds with (32)P-labeled phosphate, yield about 2000 molecules of labeled tetraphosphate per cell, this yield increasing only slightly with continued labeling. Only the tetraphosphates of adenosine and guanosine have been found in Escherichia coli, and these two are present in approximately equal amounts.     

Isopycnic centrifugation for the isolation of DNA strands coding for ribosomal RNA

Denatured DNA preparations from Escherichia coli were centrifuged to equilibrium in cesium chloride solutions. Hybridizing experiments with radioactively labeled ribosomal RNA showed that the DNA strands complementary to ribosomal RNA were distributed on the heavy side of the DNA band. By fractionating this band the DNA strands coding for ribosomal RNA may be enriched 5- to 20- fold.     

Autocatalytic synthesis of a viral RNA in vitro

Experiments with an RNA-dependent RNA polymerase ("replicase") purified from Escherichia coli infected with an RNA bacteriophage (Qbeta) demonstrate that the enzyme generates a polynucleotide of the same molecular weight as viral RNA; the "replicase" cannot distinguish the polynucleotide from its own RNA genome. By starting reactions at input ratios below the saturation levels of template to enzyme, autocatalytic kinetics of RNA increase are observed. The data are consistent with the conclusion that self-propagation of complete viral genomes is occurring in this simple system.