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Eukaryotic RNA Polymerases00:58

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RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
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Ribosome synthesis is a highly complex and coordinated process involving more than 200 assembly factors. The synthesis and processing of ribosomal components occurs not only in the nucleolus but also in the nucleoplasm and the cytoplasm of eukaryotic cells.
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The pentose sugar in DNA is deoxyribose, while in RNA the pentose sugar is ribose. The difference between the sugars is the presence of the hydroxyl group on the ribose's second carbon and a hydrogen on the deoxyribose's second carbon. The phosphate residue attaches to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms  a 5′ to 3′ phosphodiester linkage.
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The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
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Template-Independent Enzymatic RNA Synthesis.

Nilesh B Karalkar, Tatiana Kent, Taylor Tredinnick

    Biorxiv : the Preprint Server for Biology
    |October 17, 2024
    PubMed
    Summary
    This summary is machine-generated.

    Researchers developed a novel method for synthesizing RNA using a removable 3'-aminoxy blocking group and specific DNA polymerases. This template-independent enzymatic RNA synthesis (TIERS) offers a simpler, safer alternative to traditional RNA synthesis methods.

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    DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

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    Area of Science:

    • Biochemistry
    • Synthetic Biology
    • Molecular Biology

    Background:

    • Traditional RNA synthesis methods, such as phosphoramidite synthesis, often involve hazardous reagents and sensitive conditions.
    • Developing efficient and controlled methods for de novo RNA synthesis is crucial for various biotechnological applications.

    Purpose of the Study:

    • To report a novel route for preparing ribonucleoside triphosphates with a removable 3 ahydroxy (3 ahydroxy) blocking group.
    • To demonstrate template-independent enzymatic RNA synthesis (TIERS) using engineered DNA polymerases and these modified triphosphates.
    • To establish a cyclic reversible termination framework for controlled, stepwise RNA elongation.

    Main Methods:

    • Synthesis of ribonucleoside triphosphates featuring a 3 ahydroxy (3 ahydroxy) group.
    • Enzymatic assays using human DNA polymerase theta (Polθ) and mimiviral PrimPol with the modified triphosphates.
    • Demonstration of cyclic RNA synthesis through repeated nucleotide addition and 3 ahydroxy group cleavage.

    Main Results:

    • Two DNA polymerases, Polθ and PrimPol, were shown to accept the 3 ahydroxy-modified triphosphates as substrates.
    • Cleavage of the O-N bond in the 3 ahydroxy group successfully regenerated the 3 ahydroxy hydroxyl group, enabling subsequent nucleotide addition.
    • Successful stepwise addition of three ribonucleotides in three cycles using engineered Polθ demonstrated the feasibility of the TIERS process.
    • The method enables de novo RNA synthesis without hazardous solvents or sensitive reagents.

    Conclusions:

    • A novel and efficient method for template-independent enzymatic RNA synthesis (TIERS) has been established.
    • The cyclic reversible termination framework using 3 ahydroxy-modified triphosphates offers a simplified and safer alternative to phosphoramidite RNA synthesis.
    • This approach is amenable to instrument adaptation and holds promise for the de novo synthesis of RNA with defined sequences.