All Title Author
Keywords Abstract

Structural Properties of the RNA Synthesized by Glutamate Dehydrogenase for the Degradation of Total RNA

DOI: 10.4236/aer.2018.63004, PP. 29-52

Keywords: RNA Enzyme Synthesis, Nongenetic Code-Based RNA, Cloning Vector DNA, Touch-Down PCR, Frame Shift Sequence Homology

Full-Text   Cite this paper   Add to My Lib


Glutamate dehydrogenase (GDH)-synthesized RNA, a nongenetic code-based RNA is suitable for unraveling the structural constraints imposed on the regulation (transcription, translation, siRNA etc.) of metabolism by genetic code. GDH-synthesized RNAs have been induced in whole plants to knock out target mRNA populations thereby producing plant phenotypes that are allergen-free; enriched in fatty acids, essential amino acids, shikimic acid, resveratrol etc. Methods applied hereunder for investigating the structural properties of GDH-synthesized RNA included purification of GDH isoenzymes, synthesis of RNA by the isoenzymes, reverse transcription of the RNA to cDNA, sequencing of the cDNA, computation of the G+C-contents, profiling the stability through PCR amplification compared with genetic code-based DNA; and biochemical characterization of the RNAs synthesized by individual hexameric isoenzymes of GDH. Single product bands resulted from the PCR amplification of the cDNAs of GDH-synthesized RNA, whereas several bands resulted from the amplification of genetic code-based DNA. The cDNAs have wide G+C-contents (35% to 59%), whereas genetic code-based DNA has narrower G+C-contents (50% to 60%). The GDH β6 homo-hexameric isoenzyme synthesized the A+U-rich RNAs, whereas the a6, and α6 homo-hexameric isoenzymes synthesized the G+C-rich RNAs. Therefore, the RNA synthesized by GDH is different from genetic code-based RNAs. In vitro chemical reactions revealed that GDH-synthesized RNA degraded total RNA to lower molecular weight products. Therefore, GDH-synthesized RNA is RNA enzyme. Dismantling of the structural constraints imposed on RNA by genetic code liberated RNA to become an enzyme with specificity to degrade unwanted transcripts. The RNA enzyme activity of GDH-synthesized RNA is ubiquitous in cells; it is readily induced by treatment of plants with mineral nutrients etc. and may simplify experimental approaches in plant enzymology and molecular biology research projects.


[1]  Osuji, G.O., Brown, T.K. and South, S.M. (2008) Discovery of the RNA Synthetic Activity of Glutamate Dehydrogenase and Its Application in Drug Metabolism Research. The Open Drug Metabolism Journal, 2, 1-13.
[2]  Osuji, G.O. and Braithwaite, C. (1999) Signaling by Glutamate Dehydrogenase in Response to Pesticide Treatment and Nitrogen Fertilization of Peanut (Arachis hypogaea L.). Journal of Agricultural and Food Chemistry, 47, 3332-3344.
[3]  Osuji, G.O., Brown, T.K., South, S.M., Duncan, J.C., Johnson. D. and Hyllam, S. (2012) Molecular Adaptation of Peanut Metabolic Pathways to Wide Variations of Mineral Ion Composition and Concentration. American Journal of Plant Sciences, 3, 33-50.
[4]  Osuji, G.O., Brown, T.K., South, S.M., Duncan, J.C. and Johnson, D. (2011) Doubling of Crop Yield through Permutation of Metabolic Pathways. Advances in Bioscience and Biotechnology, 2, 364-379.
[5]  Osuji, G.O., Madu, W.C., Braithwaite, C., Beyene, A., Roberts, P.S., Bulgin, A. and Wright, V. (2003/4) Nucleotide-Dependent Isomerization of Glutamate Dehydrogenase in Relation to Total RNA Contents of Peanut. Biologia Plantarum, 47, 195-202.
[6]  Osuji, G.O., Brown, T.K., South, S.M., Duncan, J.C., Johnson, D. and Hyllam, S. (2012) Molecular Modeling of Metabolism for Allergen-Free Low Linoleic Acid Peanuts. Applied Biochemistry and Biotechnology, 168, 805-823.
[7]  Osuji, G.O., Duffus, E., Johnson, P., Woldesenbet, S., Weerasooriya, A., Ampim, P.A.Y., Carson, L., Jung, Y., South, S., Idan, E., Johnson, D., Clarke, D., Lawton, B. and Parks, A. (2015) Enhancement of the Essential Amino Acid Composition of Food Crop Proteins through Biotechnology. American Journal of Plant Sciences, 6, 3091-3108.
[8]  Osuji, G.O., Johnson, P., Duffus, E., Woldesenbet, S. and Kirven, J.M. (2017) Horticultural Production of Ultra-High Resveratrol Peanut. Agricultural Sciences, 8, Article ID: 80053.
[9]  Osuji, G.O., Gao, M., Carson, L., Ampim, P., Weerasooriya, A., Johnson, P., Duffus, E., Woldesenbet, S., Kirven, W.E., Johnson, D. and Clarke, D. (2017) Biotechnological Production of Shikimate-Based Antioxidant Accumulation in Phyla dulcis. The Natural Products Journal, 7, 104-111.
[10]  Cech, T.R. (1986) RNA as an Enzyme. Scientific American, 255, 64-75.
[11]  Klahre, U., Crete, P., Leuenberger, S.A., Iglesias, V.A. and Meins, F. (2002) High Molecular Weight RNAs and Small Interfering RNAs Induce Systemic Postrranscriptional Gene Silencing in Plants. Proceedings of the National Academy of Sciences of USA, 10, 1073-1078.
[12]  Agrawal, N., Dasaradhi, P.V.N., Mohamed, A., Malhotra, P., Bhtnagar, R.K. and Mukherjea, S.K. (2003) RNA Interference: Biology, Mechanism, and Applications. Microbiology and Molecular Biology Reviews, 67, 657-685.
[13]  Di Chiacchio, L., Sloma, M.F. and Mathews, D.H. (2016) Access Fold: Predicting RNA-RNA Interactions with Consideration for Competing Self-Structure. Bioinformatics, 32, 1033-1039.
[14]  Meyer, I.M. (2008) Predicting RNA-RNA Interactions. Current Opinion in Structural Biology, 18, 387-393.
[15]  Clowney, L., Jain, C.S., Srinivasan, A.R., Westbrook, J., Olson, W.K. and Berman, H.M. (1996) Geometric Parameters in Nucleic Acids. Nitrogenous Bases. Journal of American Chemical Society, 118, 509-518.
[16]  Chan, C.Y., Carmack, C.S., Long, D.D., Maliyekkel, A, Shao, Y., Roninson, I.B. and Ding, Y. (2009) A Structural Interpretation of the Effect of GC-Content on Efficiency of RNA Interference. BMC Bioinformatics, 10, S33.
[17]  Mann, S. and Chen, Y.P. (2010) Bacterial Genomic G+C Composition-Eliciting Environmental Adaptation. Genomics, 95, 7-15.
[18]  Smarda, P., Bures, P., Horova, L., Leitch, I.J., Mucina, L., Tichy, G.V. and Rotreklova, O. (2014) Ecological and Evolutionary Significance of Genomic GC Content Diversity in Monocots. Proceedings of the National Academy of Sciences, 111, E4096-E4102.
[19]  Liu, L., Li, Q., Lin, H. and Zou, Y. (2013) The Effect of Regions Flanking Target Site on siRNA Potency. Genomics, 102, 215-222.
[20]  Oliver, J.L. and Marin, A. (1996) A Relationship between GC Content and Coding-Sequence Length. Journal of Molecular Evolution, 43, 216-223.
[21]  Yakovchuk, P., Protozanova, E. and Frank-Kamenetskii, M.D. (2006) Base-Stacking and Base-Pairing Contributions into Thermal Stability of the DNA Double Helix. Nucleic Acids Research, 34, 564-574.
[22]  Krutinin, G.G., Krutinina, E.A., Kamzolova, S.G. and Osypov, A.A. (2015) Bacteriophage λ: Electrostatic Properties of the Genome and Its Elements. Molecular Biology, 49, 339-347.
[23]  Cuervo, A., Dans, P.D., Carrascosa, J.L., Orozco, M., Gomila, G. and Fumagalli, L. (2014) Direct Measurement of the Dielectric Polarization Properties of DNA. Proceedings of the National Academy of Sciences, 111, E3624-E3630.
[24]  Vologodskii, A. and Cozzarelli, N. (1995) Modelling Long-Range Electrostatic Interactions in DNA. Biopolymers, 35, 289-296.
[25]  Osuji, G. and Brown, T. (2007) Role of the RNA Synthesized by Glutamate Dehydrogenase in the Coordinate Regulation of Metabolic Processes. The ICFAI Journal of Biotechnology, 1, 37-48.
[26]  Osuji, G.O. and Madu, W.C. (2015) Glutamate Dehydrogenase. In: D’Mello, J.P.F., Ed., Amino Acids in Higher Plants, CABI Publishers, Oxfordshire and Boston, 1-29.
[27]  Grierson, D., Slater, J. and Tucker, G.A. (1985) The Appearance of Polygalacturonase mRNA in Tomatoes. Planta, 163, 263-271.
[28]  Loyola-Vargas, V.M. and De Jimenez, E.S. (1984) Differential Role of Glutamate Dehydrogenase in Nitrogen Metabolism of Maize Tissue. Plant Physiology, 76, 536-540.
[29]  Osuji, G.O., Konan, J. and M’Mbijjewe, G. (2004) RNA Synthetic Activity of Glutamate Dehydrogenase. Applied Biochemistry and Biotechnology, 119, 209-228.
[30]  Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic Local Alignment Search Tool. Journal of Molecular Biology, 215, 403-410.
[31]  Tatusova, T. and Madden, T.L. (1999) Blast 2 Sequences—A New Tool for Comparing Protein and Nucleotide Sequences. FEMS Microbiology Letters, 174, 247-250.
[32]  Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K. and Mattick, J.S. (1991) “Touchdown” PCR to Circumvent Spurious Priming during Gene Amplification. Nucleic Acids Research, 19, 4008.
[33]  Erlich, H.A., Gelfand, D. and Sninsky, J.J. (1991) Recent Advances in the Polymerase Chain Reaction. Science, 252, 1643-1651.
[34]  Kramer, M.F. and Coen, D.M. (2001) Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization. Current Protocols in Toxicology, 3, 1-14.
[35]  Korbie, D.J. and Mattick, J.S. (2008) Touchdown PCR for Increased Specificity and Sensitivity in PCR Amplification. Nature Protocols, 3, 1452-1456.
[36]  Glemin, S., Clement, Y., David, J. and Ressayre, A. (2014) GC Content Evolution in Coding Regions of Angiosperm Genomes: A Unifying Hypothesis. Trends in Genomes, 30, 263-270.
[37]  Vinogradov, A.E. (2003) DNA Helix: The Importance of Being GC-Rich. Nucleic Acids Research, 31, 1838-1844.
[38]  Meier-Kolthoff, J.P., Klenk, H. and Goker, M. (2014) Taxonomic Use of DNA G+C Content and DNA-DNA Hybridization in the Genomic Age. International Journal of Systematic Environmental Microbiology, 64, 352-356.
[39]  Bernadi, G. (1989) The Isochore Organization of the Human Genome. Annual Review of Genetics, 23, 637-661.
[40]  Frank-Kamenetskii, M.D. (1987) How the Double Helix Breaths. Nature, 328, 17-18.
[41]  Krueger, U., Bergauer, T., Kaufmann, B., Wolter, I., Pilk, S, Heider-Fabian, M., Kirch, S., Artz-Oppitz, C., Isselhorst, M. and Konrad, J. (2007) Insights into Effective RNAi Gained from Large-Scale siRNA Validation Screening. Oligonucleotides, 17, 237-250.
[42]  Stein, C.A. (1998) How to Design an Antisense Oligodeoxynucleotide Experiment: A Consensus Approach. Antisense and Nucleic Acid Drug Development, 8, 129-132.
[43]  Cech, T.R., Zaug, A.J. and Grabowski, P.J. (1981) In Vitro Splicing of the rRNA Precursor of Tetrahymena: Involvement of a Guanosine Nucleotide in the Excision of the Intervening Sequence. Cell, 27, 487-496.
[44]  Eckstein, F. and Lilley, D.M.J. (1996) Catalytic RNA. In: Nucleic Acids and Molecular Biology, Vol. 10, Springer-Verlag, Berlin.
[45]  Osuji, G.O., Brown, T.K. and South, S.M. (2010) Optimized Fat and Cellulosic Biomass Accumulation in Peanut through Biotechnology. International Journal Biotechnology and Biochemistry, 6, 445-476.
[46]  Osuji, G.O., Weerasooriya, A., Ampim, P.A.Y., Carson, L., Johnson, P., Jung, Y., Duffus, E., Woldesenbet, S., South, S., Idan, E., Johnson, D., Clarke, D., Lawton, B., Parks, A. and Fares, A. (2015) Molecular Regulation of the Metabolic Pathways of the Medicinal Plants: Phyla dulcis. American Journal of Plant Sciences, 6, 1717-1726.
[47]  Takahashi, S., Yeo, Y., Greenhagen, B.T., McMullin, T., Song, L., Maurina-Brunker, J., Rosson, R., Noel, J.P. and Chappell, J. (2007) Metabolic Engineering of Sesquiterpene Metabolism in Yeast. Biotechnology and Bioengineering, 97, 170-181.
[48]  Liu, S., Lv, Y., Wang, X.R., Li, L.M., Hu, B. and Li, L. (2014) Cloning and Expression Analysis of cDNAs Encoding ABA 8’-Hydrolase in Peanut Plants in Response to Osmotic Stress. PLoS ONE, 9, e97025.
[49]  Chen, N., Yang, Q., Su, M., Pan, L., Chi, X., Chen, M., He, Y., Yang, Z., Wang, T. and Yu, S. (2012) Cloning of Six EFR Family Transcription Factors Genes from Peanut and Analysis of Their Expression during Abiotic Stress. Plant Molecular Biology Reporter, 30, 1415-1425.
[50]  Strobel, S.A. and Cochrane, J.C. (2007) RNA Catalysis: Ribozymes, Ribosomes and Riboswitches. Current Opinion in Chemical Biology, 11, 636-643.
[51]  Lilley, D.M.J. (2011) Mechanisms of RNA Catalysis. Philosophical Transactions of the Royal Society B: Biological Sciences, 366, 2910-2917.
[52]  Miyagishi, M. and Taira, K. (2005) siRNA Becomes Smart and Intelligent. Nature Biotechnology, 23, 946-947.
[53]  Jagla, B., Aulner, N., Kelly, P.D., Song, D., Volchuk, A., Zatorski, A., Shum, D., Mayer, T., De Anglis, D.A., Ourfelli, O., Rutishauser, U. and Rothman, J.E. (2005) Sequence Characteristics of Functional siRNAs. RNA, 11, 864-872.
[54]  Larsson, E., Sander, C. and Marks, D. (2010) mRNA Turnover Rate Limits siRNA and microRNA Efficacy. Molecular Systems Biology, 6, 433.
[55]  Hong, S.W., Jang, Y., Kim, S., Li, C.J. and Lee, D. (2014) Target Gene Abundance Contributes to the Efficiency of siRNA-Mediated Gene Silencing. Nucleic Acid Therapeutics, 24, 192-198.


comments powered by Disqus