Ribonucleotide: Definition, Overview, & Applications

Ribonucleotide: Definition, Overview, & Applications

Ribonucleotide Overview

Nucleotides are the building blocks of nucleic acids — one of the four essential groups of biomolecules among proteins, carbohydrates, and amino acids.

The basic skeleton of nucleotide (or nucleoside phosphate) is made of pentose sugar, phosphate, and a nitrogenous base (purine or pyrimidine). And, based on the type of pentose sugar the nucleotide contains, it’s of two types: ribonucleotide and deoxyribonucleotide.

Ribonucleotide is a nucleotide having ribose as its pentose sugar. Whereas deoxyribonucleotide contains deoxyribose sugar.

The ribonucleotide molecule acts as a precursor for nucleic acid synthesis. It can be transformed into deoxyribose sugar after the reduction reaction facilitated by ribonucleotide reductase (RNR) — an enzyme first discovered in E.coli (Escherichia coli) and has a catalytic mechanism in ribonucleotide reduction.

The ribonucleotide is mainly used for the synthesis of RNA. Whereas deoxyribonucleotide is used in the DNA synthesis process.

The nitrogenous bases of ribonucleotides are grouped into two groups: purine and pyrimidine. They consist of four molecules, which include adenine (A), guanine (G), cytosine (C), and uracil (U). The difference between DNA and RNA developing nucleotides is the presence of thymine, which is only involved in the DNA replication process and not in RNA synthesis.

The presence and absence of phosphate groups in the ribonucleotide structure change the whole chemistry of the biomolecule. In absence of a phosphate group, the molecule is known as ribonucleoside rather than ribonucleotides. Also, based on the number of phosphates, ribonucleotides can be monophosphates (having one phosphate group), diphosphates (having two phosphate groups), and triphosphates (having three phosphate groups).

Figure: Basic structure of ribonucleotides.

Ribonucletides have a myriad of functions in organisms, ranging from DNA replication, transcription (the process of mRNA synthesis), DNA repair, and gene expression to acting as a substrate for ATP (adenosine triphosphate) and AMP (adenosine monophosphate) production and metabolic regulation.

In this article, we will review more on the functions of the ribonucleotides, their synthesis, and their applications in a range of biotech, molecular biology, and biochemistry labs.

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How Does Ribonucleotide Work?

Ribonucleotides have a major role in RNA synthesis. Several ribonucleotide residues joined together in an RNA helix via phosphodiester bonds. A backbone of alternating phosphate and pentose residues is created by linking the 5′-phosphate group of one nucleotide to the 3′-hydroxyl group of the next nucleotide.  

The 3′-hydroxyl group of the last ribonucleotide of the chain acts as a nucleophile and attacks the 5′-triphosphate of the incoming ribonucleotide, releasing pyrophosphate as a byproduct. The reaction is facilitated by the enzyme RNA polymerase.

Ribonucleotide Synthesis

Ribonucleotide synthesis in organisms either occurs through a de novo pathway or the salvage pathway. In the de novo synthesis, the biosynthesis of purines and pyrimidines occurs through the precursors, like ribose-5-phosphates, CO2, amino acids, NH3, and other small molecules.

The enzymatic reactions involved in the process are facilitated by many enzymes, such as ribose-phosphate diphosphokinase (PRPS1), GAR synthetase, GAR transformylase, cytidylate synthetase, and dihydroorotase dehydrogenase.

Figure: A schematic diagram of the de novo pathway of pyrimidine synthesis.

Ribonucleotide Reductase (RNR)

Ribonucleotide reductase (RNR) is a ubiquitous enzyme found in all eukaryotes, and some prokaryotes, archaea, and viruses. It is grouped into two classes: Classes I RNR and Class II RNR.The class I enzyme is composed of two alpha and beta tetramers. The alpha subunit is the catalytic subunit, which contains redox-active cysteines (performing both oxidation and reduction), active site, and allosteric binding sites. The beta subunit of the enzyme contains a di-iron cluster that reacts with dioxygen to form a tyrosyl radical and a di-iron(III) cluster.

Figure: The crystal structure (heterooctamer) of the ribonucleotide reductase (RNR) of E.coli.

The mammalian ribonucleotide reductase has two identical large subunits, which is also known as catalytic subunit, and two identical small subunits that are known as regulatory subunits

Some enzymatic reactions occur by the association between RNR I and RNR II. The active site of RNR I contain dithiol groups, and RNR II has a ferric center and the tyrosyl radical. Some other residues involved in the active site of RNR II include tyrosine, tryptophan, and aspartate. Studies like site-directed mutagenesis have proved the roles of these metal ions in the transfer of radicals to the active site of the enzyme.

What is Ribonucleotide Used For?

Ribonucleotides have a range of cellular functions in organisms. It plays a key role in cell regulation, transcription, genomic and mitochondrial DNA replication, cell cycle-specific proteolysis in mammalian cells, and cell signaling.

rNTP Removal

Ribonucleotides are present at much higher concentrations than deoxyribonucleotides in organisms. Thus, DNA polymerases need to discriminate between both of them to ensure the accuracy of base incorporation in organisms’ genomes.

The Y family of ribonucleotides has active sites that have substrate specificity to identify and eliminate the ribonucleotides. However, the system is not perfect, and some ribonucleotides still get incorporated into DNA, which is taken care of by the repair enzymes to prevent genomic instability.

Deoxyribonucleotide Synthesis

Ribonucleotides act as a precursor for the synthesis of deoxyribonucleotides, such as dATP, dTTP, dGTP, and dCTP. The reaction is catalyzed by the enzyme ribonucleotide reductase (RNR).

Other than ribonucleotide reductase (RNR), the two other enzymes required for DNA replication are thioredoxin and thioredoxin reductase. At first, the ribonucleotide diphosphates (such as ADP and GDP) are converted to deoxyribonucleoside diphosphate (dNDPs, such as dADP and dGDP) by the enzyme thioredoxin and ribonucleotide reductase (RNR).

Then, the resulting deoxyribonucleotides are phosphorylated by nucleoside diphosphate kinases to form dNTPs (or deoxyribonucleoside triphosphates), such as dATP and dGTP.

Once dATP is synthesized, it binds to ribonucleotide reductase (RNR), which decreases enzymatic catalysis. Thus, dATP acts as an inhibitor of the RNR enzymatic activity. However, the inhibition is reversed as ATP binds to the enzyme. Moreover, the enzymatic action of RNR can also be interfered with using the drugs hydroxyurea and Motexafin gadolinium.

What Industries Use Ribonucleotide?

Ribonucleotides have extensive applications in a range of in vivo and in vitro processes, such as transcription, RNA amplification, ligation, phosphorylation, creating site-directed mutations, and siRNA synthesis for many research purposes.


Because of the massive involvement of ribonucleotides in a range of metabolic processes, biochemistry labs and industries use them to synthesize rNTPs, dNTPs, RNA, and DNA molecules. It helps to study and understand the metabolic processes and pathological relations of certain molecules and pathways.

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Ribonucleotides are a basic building block of RNA. Each unit is joined by a phosphodiester bond to form an RNA helix. The basic structure of ribonucleotide is composed of ribose sugar, nitrogenous base, and phosphate. Based on the bases, ribonucleotides are uridine (with base uracil), cytidine (cytosine base), adenosine (adenine base), and guanosine (guanine base).

The molecule has wide applications in many in vivo and in vitro processes, which include DNA replication, transcription, mitochondrial DNA synthesis, and cell signaling. Thus, it’s used in many life sciences and biochemistry labs to conduct several research studies.

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