Walter R. Farkas

Depolymerization of ribonucleic acid by plumbous ion

Abstract 1. 1.1 mM Pb2+ catalyzed the depolymerization of RNA at 37° in the regions of neutral pH. The reaction proceeded most rapidly at pH 7.5 but the pH optimum was dependent on Pb2+ concentration and was 8.5 at 0.125 mM Pb2+. The products of the reaction consisted primarily of oligonucleotides phosphorylated at the 3′ terminus. 2. 2. Poly (I) was degraded more rapidly than poly (C). Poly (A) and poly (U) were attacked at similar rates but more rapidly than poly (I). 2′,3′-Cyclic ribonucleotides and dinucleoside phosphates were not attacked. 3. 3. Secondary structure in the polynucleotides protected against depolymerization by Pb2+ as was shown by the decreased rate at which complimentary polynucleotides were degraded. The poly (A)-poly (U) hybrid was attacked at one third the rate of poly (A) or poly (U) alone. The poly (I)-poly (C) complex was refractory to Pb2+ catalyzed depolymerization. 4. 4. No other cation tested was similarly effective in promoting RNA degradation.

Substrate and inhibitor specificity of tRNA-guanine ribosyltransferase

We have tested as inhibitors or substrates of tRNA-guanine ribosyltransferase (EC 2.4.2.29) a number of compounds, including derivatives of 7-deazaguanine, pteridines, purines, pyrimidines and antimalarials. Virtually all purines and pteridines that are inhibitors or substrates of the rabbit reticulocyte enzyme have an amino nitrogen at the 2 position. In addition the 9 position and the oxygen at the 6 position may be important for recognition by the enzyme. Saturation of the double bond in the cyclopentenediol moiety of queuine reduces substrate activity and queuine analogs that lack the cyclopentenediol moiety, such as 7-deazaguanine and 7-aminomethyl-7-deazaguanine, are relatively poor substrates for the enzyme. While adenosine is not an inhibitor, neplanocin A (an adenosine analog in which a cyclopentenediol replaces the ribose moiety) is a poor inhibitor. The incorporation of 7-aminomethyl-7-deazaguanine into the tRNA of L-M cells results in a novel chromatographic form of tRNAAsp, indicating that L-M cells cannot modify this Q precursor (in Escherichia coli) to queuosine. The specific incorporation of 7-deazaguanine and 8-azaguanine into tRNA by L-M cells also results in novel chromatographic forms of tRNAAsp. With intact L-M cells, the enzyme-catalyzed insertion into tRNA of queuine, dihydroqueuine, 7-aminomethyl-7-deazaguanine, or 7-deazaguanine is irreversible, while guanine or 8-azaguanine incorporation is reversible; suggesting that it is the substitution of C-7 for N-7 which prevents the reversible incorporation of queuine into tRNA.

Guanylation of transfer RNA by rabbit reticulocytes.

Abstract 1. Incubation of rabbit reticulocytes in the presence of [14C]guanine brings about incorporation of this compound into reticulocyte tRNA. 2. The incorporation of [14C]guanine into tRNA is not due to terminal addition at either the 3′ or 5′ end. It appears that the guanine is incorporated into an internal position of the polynucleotide. 3. Freon reverse phase chromatography of the guanylated RNA indicates that only two species of reticulocyte tRNA may be guanylated. 4. Histidine has been identified as the amino acid accepted by guanylated tRNA. 5. Guanylation is not inhibited by actinomycin D or puromycin. It is weakly inhibited by cycloheximide and 8 azaguanine.

Effects of plumbous ion on some functions of transfer RNA.

Abstract Plumbous ion has been shown to catalyze the degradation of RNA, and the effects of lead-catalyzed depolymerization on some of the functions of tRNA has been investigated. Exposure to Pb2+ brings about a rapid decrease but not a complete loss of amino acid acceptance capacity. The tRNA molecules responsible for this residual activity have not been degraded and represent a class of molecules that are resistant to Pb2+. The ratio of susceptible to resistant tRNA increases when the temperature is raised. The effect of varying the Pb2+ concentration was studied, and there is a threshold Pb2+ concentration below which the tRNA is not inactivated. The ability of Pb2+ depolymerized tRNA to bind to ribosomes in response to its appropriate codon is impaired to a greater extent than the ability of the tRNA to accept amino acids. The Pb2+ concentration at which binding capacity is lost is at least two orders of magnitude above the level at which protein synthesis is inhibited in mammalian erythroid cells.

Effect of plumbous ion on messenger RNA

The effects of Pb2+, a potent catalyst for the depolymerization of RNA have been studied on brome mosaic virus (BMV) RNA, rabbit globin m-RNA and polyuridylic acid. After exposure of these natural and synthetic messengers to a sufficiently high concentration of Pb2+, they all lost their ability to stimulate amino acid incorporation in cell-free protein-synthesizing systems. There were differences in the susceptibilities of the messengers; gloing the m-RNA for 40 min revealed that there was a threshold Pb2+ concentration below which no loss of m-RNA activity was observed. The threshold concentration was considerably greater than the Pb2+ concentration at which protein synthesis is inhibited in reticulocytes and overt symptoms of plumbism are observed. However, when m-RNA were incubated for an extended period (24 h), even with sub-threshold concentrations of Pb2+, there was destruction of messenger function and globin m-RNA was more susceptible than BMV-RNA. Also the susceptibility of m-RNA to Pb2+ is temperature-dependent, which would indicate that m-RNA, like t-RNA, exists as a population of molecules in different conformational states that are not readily interconvertible.

Partial purification and properties of the reticulocyte guanylating enzyme.

The guanylating enzyme which catalyzes the insertion of a guanine residue into one of the isoacceping tRNAHis of rabbit reticulocytes has been purified approximately one-hundred fold. It is free of nuclease activity. The enzyme does not catalyze the replacement of inserted radioactive guanine by unlabeled guanine, indicating that the reaction is irreversible. We have separated the histidyl-tRNA of reticulocytes into three isoacceptors. Previous work showed that the last histidyl-tRNA to elute from RPC-5 columns was the product of the guanylation reaction. This reports shows that the same late-eluting peak also contains the substrate for the guanylating enzyme, indicating that the guanine insertion reaction is chromatographically silent. The isoaccepting tRNAHis that is the substrate for the guanylating enzyme does not contain the hypermodified base known as Q. It is the other major reticulocyte tRNAHis that coantains Q, showing that at least in the reticulocyte the role of the guanylating enzyme is not the conversion of the Q form of tRNA to the homogeneic G form. The purified enzyme does not insert any base other than guanine into tRNA.

tRNA-guanine transglycosylase and guanine-accepting transfer RNAs of Drosophila melanogaster

tRNA-guanine transglycosylase replaces a base in tRNA with guanine and uses free guanine as the substrate. This enzyme was obtained from adult Drosphila melanogaster and characterized as follows: Km for guanine is 2.8 × 10−7M; pH optimum is 7.4. The enzyme activity is present in third instar larvae and adult flies but is absent in late pupae. When Drosphila tRNAs were guanylated with the comparable enzyme from rabbit reticulocytes, at least four species of tRNA could be demonstrated to have incorporated radioactive guanine. The role for this enzyme appears to be to exchange queuine into tRNA and thus replace a guanine in the anticodon of tRNAs for Asn, Asp, His, and Tyr. In the absence of queuine it will catalyze the exchange reaction described above.

The guanylation of transfer RNA: An enzymatic reaction

Abstract 1. 1. Reticulocytes of rabbits, sheep and humans but not those of rats or mice incorporate guanine into tRNA after the transcription of the polynucleotide chain is completed. 2. 2. The reaction is heat labile and is inhibited by iodoacetate, iodoacetamide and 8-azaguanine. 3. 3. No other purine is similarly incorporated into tRNA. 4. 4. The incorporated guanine is recovered as a single oligonucleotide after digestion of the guanylated tRNA with T1 ribonuclease and the incorporated guanine is present at the 3′ end of that oligonucleotide. 5. 5. The rate of guanylation in reticulocytes is concentration dependent within limits; and an apparent K m for guanylation of 2.2 · 10−5 M has been obtained. The K m for guanosine is 5.8 · 10−6 M. 6. 6. These findings point to an enzymatic mechanism for guanylation.

Secretion of ceruloplasmin by a human clear cell carcinoma maintained in nude mice

Ceruloplasmin is the best known but least understood copper protein. Studies preliminary to investigating the control of ceruloplasmin synthesis have utilized a human renal cell carcinoma maintained in nude mice for 73 passages over a 5-year period. In vitro cultures of these cells were accomplished and the mRNAs were extracted prior to microinjection into Xenopus oocytes. The media examined by SE-HPLC and immunological techniques demonstrated that (1) after in vitro culture, ceruloplasmin was secreted as an uncleaved polypeptide chain with a MW of 135,000; (2) the translational product of ceruloplasmin mRNA injected into Xenopus oocytes was cleaved into fragments with MWs of 110,000, 67,000, and 50,000. The results indicate that mRNA for human ceruloplasmin can be obtained to serve as a template for the synthesis of a cDNA probe to investigate the control of human ceruloplasmins synthesis.

Queuine, The Q-Containing tRNAs and the Enzymes Responsible for Their Formation

Abstract Queuine was first observed by Goodman et al. during studies on suppression of nonsense mutations in E. coli (1). This report was soon confirmed by Doctor et al.. (2). In that study the molecule responsible for suppression of nonsense was found to be a mutated tRNATYr. While the suppressor tRNATYr contained cytidine in the first position of the anticodon, wild-type tRNATYr contained a modified derivative of guanine in the first position of the anticodon. The modified nucleoside was called G∗ and had an ultraviolet absorption spectrum very similar to guanosine. They reported that G∗ had an additional basic group with a pKa between 5 and 6. T1 ribonuclease, which cleaves RNA to form oligonucleotides ending in G at their 3′ end, did not cleave the phosphodiester bond on the 3′ side of G∗. The tRNATYr containing G∗ had the same codon recognition properties as if unmodified guanine was present, leading the authors to speculate that the modification was on the imidazole or non-base pairing part of the p...