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RNA Substrate (rna + substrate)
Selected AbstractsAn allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activitiesFEBS JOURNAL, Issue 11 2010Dazhi Jiang A series of RNA-cleaving or DNA-cleaving DNAzymes have been obtained by in vitro selection. However, engineering an allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activities is very challenging. We used an in vitro -selected pistol-like (PL) DNAzyme as a DNA scaffold for designing a DNAzyme with dual catalytic activities. We prepared the 46-nucleotide DNAzyme with DNA-cleaving activity (PL DNAzyme), and then grafted the deoxyribonucleotide residues from an 8,17 variant DNAzyme into the region of stem,loop I and the catalytic core of the PL DNAzyme scaffold. This deoxyribonucleotide residue grafting resulted in a DNAzyme with dual RNA-cleaving and DNA-cleaving activities (DRc DNAzyme). Drc DNAzyme has properties different from those of the original PL DNAzyme, including DNA cleavage sites and the required metal ion concentration. Interestingly, the RNA substrate and RNase A can act as effectors to mediate the DNA cleavage. Our results show that RNA-cleaving and DNA-cleaving activities simultaneously coexist in DRc DNAzyme, and the DNA cleavage activity can be reversibly regulated by a conformational transition. [source] A mutagenic analysis of the RNase mechanism of the bacterial Kid toxin by mass spectrometryFEBS JOURNAL, Issue 17 2009Elizabeth Diago-Navarro Kid, the toxin of the parD (kis, kid) maintenance system of plasmid R1, is an endoribonuclease that preferentially cleaves RNA at the 5, of A in the core sequence 5,-UA(A/C)-3,. A model of the Kid toxin interacting with the uncleavable mimetic 5,-AdUACA-3, is available. To evaluate this model, a significant collection of mutants in some of the key residues proposed to be involved in RNA binding (T46, A55, T69 and R85) or RNA cleavage (R73, D75 and H17) were analysed by mass spectrometry in RNA binding and cleavage assays. A pair of substrates, 5,-AUACA-3,, and its uncleavable mimetic 5,-AdUACA-3,, used to establish the model and structure of the Kid,RNA complex, were used in both the RNA cleavage and binding assays. A second RNA substrate, 5,-UUACU-3, efficiently cleaved by Kid both in vivo and in vitro, was also used in the cleavage assays. Compared with the wild-type protein, mutations in the residues of the catalytic site abolished RNA cleavage without substantially altering RNA binding. Mutations in residues proposed to be involved in RNA binding show reduced binding efficiency and a corresponding decrease in RNA cleavage efficiency. The cleavage profiles of the different mutants were similar with the two substrates used, but RNA cleavage required much lower protein concentrations when the 5,-UUACU-3, substrate was used. Protein synthesis and growth assays are consistent with there being a correlation between the RNase activity of Kid and its inhibitory potential. These results give important support to the available models of Kid RNase and the Kid,RNA complex. [source] Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domainsFEBS JOURNAL, Issue 2 2007Anne R. Karow RNA helicases mediate structural rearrangements of RNA or RNA,protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis,Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication. [source] RNase P RNA-mediated cleavageIUBMB LIFE, Issue 3 2009Leif A. Kirsebom Abstract Metal(II)-induced hydrolysis of RNA produce products with 5,-hydroxyls and 2,;3,-cyclic phosphates at the ends. Ribozymes are RNA molecules that act as catalysts. Some ribozymes that cleave RNA also generate 5,-hydroxyls and 2,;3,-cyclic phosphates whereas others produces 5,-phosphates and 3,-hydroxyls at the ends of the cleavage products. RNase P is an essential endoribonuclease involved in RNA processing. The catalytic RNA subunit of RNase P is a trans-acting ribozyme that cleaves various RNA substrates in vitro generating 5,-phosphates and 3,-hydroxyls as cleavage products. The activity depends on the presence of metal(II) ions such as Mg2+. RNase P RNA has therefore to facilitate a nucleophilic attack that generates the correct product ends and prevent metal(II)-induced hydrolysis of the RNA substrate. In this review, we will discuss our current understanding of the interactions between RNase P RNA and its substrate, role of specific residues with respect to catalysis and positioning of functionally important Mg2+ at and in the vicinity of the cleavage site that ensures that products with correct ends are generated. Moreover, we will discuss the composition of RNase P and its RNA subunit in an evolutionary perspective. © 2009 IUBMB IUBMB Life, 61(3):189,200, 2009 [source] Development of fully functional proteins with novel glycosylation via enzymatic glycan trimmingJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2009Melinda L. Toumi Abstract Recombinant glycoproteins present unique challenges to biopharmaceutical development, especially when efficacy is affected by glycosylation. In these cases, optimizing the protein's glycosylation is necessary, but difficult, since the glycan structures cannot be genetically encoded, and glycosylation in nonhuman cell lines can be very different from human glycosylation profiles. We are exploring a potential solution to this problem by designing enzymatic glycan optimization methods to produce proteins with useful glycan compositions. To demonstrate viability of this new approach to generating glycoprotein-based pharmaceuticals, the N -linked glycans of a model glycoprotein, ribonuclease B (RNase B), were modified using an ,-mannosidase to produce a new glycoprotein with different glycan structures. The secondary structure of the native and modified glycoproteins was retained, as monitored using circular dichroism. An assay was also developed using an RNA substrate to verify that RNase B had indeed retained its function after being subjected to the necessary glycan modification conditions. This is the first study that verifies both activity and secondary structure of a glycoprotein after enzymatic glycan trimming for use in biopharmaceutical development methods. The evidence of preserved structure and function for a modified glycoprotein indicates that extracellular enzymatic modification methods could be implemented in producing designer glycoproteins. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2581,2591, 2009 [source] RNase P RNA-mediated cleavageIUBMB LIFE, Issue 3 2009Leif A. Kirsebom Abstract Metal(II)-induced hydrolysis of RNA produce products with 5,-hydroxyls and 2,;3,-cyclic phosphates at the ends. Ribozymes are RNA molecules that act as catalysts. Some ribozymes that cleave RNA also generate 5,-hydroxyls and 2,;3,-cyclic phosphates whereas others produces 5,-phosphates and 3,-hydroxyls at the ends of the cleavage products. RNase P is an essential endoribonuclease involved in RNA processing. The catalytic RNA subunit of RNase P is a trans-acting ribozyme that cleaves various RNA substrates in vitro generating 5,-phosphates and 3,-hydroxyls as cleavage products. The activity depends on the presence of metal(II) ions such as Mg2+. RNase P RNA has therefore to facilitate a nucleophilic attack that generates the correct product ends and prevent metal(II)-induced hydrolysis of the RNA substrate. In this review, we will discuss our current understanding of the interactions between RNase P RNA and its substrate, role of specific residues with respect to catalysis and positioning of functionally important Mg2+ at and in the vicinity of the cleavage site that ensures that products with correct ends are generated. Moreover, we will discuss the composition of RNase P and its RNA subunit in an evolutionary perspective. © 2009 IUBMB IUBMB Life, 61(3):189,200, 2009 [source] Broadening the mission of an RNA enzymeJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Michael C. Marvin Abstract The "RNA World" hypothesis suggests that life developed from RNA enzymes termed ribozymes, which carry out reactions without assistance from proteins. Ribonuclease (RNase) P is one ribozyme that appears to have adapted these origins to modern cellular life by adding protein to the RNA core in order to broaden the potential functions. This RNA-protein complex plays diverse roles in processing RNA, but its best-understood reaction is pre-tRNA maturation, resulting in mature 5' ends of tRNAs. The core catalytic activity resides in the RNA subunit of almost all RNase P enzymes but broader substrate tolerance is required for recognizing not only the diverse sequences of tRNAs, but also additional cellular RNA substrates. This broader substrate tolerance is provided by the addition of protein to the RNA core and allows RNase P to selectively recognize different RNAs, and possibly ribonucleoprotein (RNP) substrates. Thus, increased protein content correlated with evolution from bacteria to eukaryotes has further enhanced substrate potential enabling the enzyme to function in a complex cellular environment. J. Cell. Biochem. 108: 1244,1251, 2009. © 2009 Wiley-Liss, Inc. [source] Consequences of RNase E scarcity in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 4 2002Chaitanya Jain Summary The endoribonuclease RNase E plays an important role in RNA processing and degradation in Escherichia coli. The construction of an E. coli strain in which the cellular concentration of RNase E can be precisely controlled has made it possible to examine and quantify the effect of RNase E scarcity on RNA decay, gene regulation and cell growth. These studies show that RNase E participates in a step in the degradation of its RNA substrates that is partially or fully rate-determining. Our data also indicate that E. coli growth requires a cellular RNase E concentration at least 10,20% of normal and that the feedback mecha-nism that limits overproduction of RNase E is also able to increase its synthesis when its concentration drops below normal. The magnitude of the in-crease in RNA longevity under conditions of RNase E scarcity may be limited by an alternative pathway for RNA degradation. Additional experiments show that RNase E is a stable protein in E. coli. No other E. coli gene product, when either mutated or cloned on a multicopy plasmid, seems to be capable of compensating for an inadequate supply of this essential protein. [source] Stress-related RNase PR-10c is post-translationally modified by glutathione in birchPLANT CELL & ENVIRONMENT, Issue 6 2002K. M. Koistinen Abstract The PR-10c (previously termed as Bet v 1-Sc3) protein of birch belongs to the family of intracellular pathogenesis-related proteins. The high-performance liquid chromatography electrospray ionization ion trap mass spectrometry (HPLC-ESI-MS) analysis of PR-10c-His fusion protein, produced in Escherichia coli, revealed three major peaks and masses. Enzymatic digestions and HPLC-ESI-MS and matrix assisted laser desorption/ionization , time of flight mass spectrometry (MALDI-TOF-MS) analyses of each fraction indicated that PR-10c-His protein is post-translationally modified by carbamylation and S-glutathiolation. Carbamylation was localized into the N-terminal end of PR-10c-His and does not represent a biologically significant modification. The possible nuclease activity of PR-10c was analysed with S-glutathiolated and reduced fractions of PR-10c-His fusion protein. Both forms of PR-10c-His as well as the dimeric form of the protein possess RNase activity which is capable of digesting different RNA substrates. None of the fractions showed activity against single- or double-stranded DNA. The MALDI-TOF-MS analysis of PR-10c polypeptide extracted from zinc-exposed birch roots showed that the protein is post-translationally modified by glutathione (, -Glu-Cys-Gly) also in vivo. The S-glutathiolated cysteine residue of PR-10c is not conserved among Bet v 1 homologous proteins and is also unique in the PR-10 family. As far as we know this is the first observation of S-glutathiolation in plants, or any post-translational modification in the PR-10 family of proteins. [source] | |||||||||||||