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An ATP-dependent RNA ligase catalyzes RNA ligation using ATP to activate the joining chemistry. For the activity EC6.5.1.3, a $3^{\prime }$ hydroxyl group is joined to a $5^{\prime }$ phosphate, yielding a phosphodiester bond, adenosine monophosphate and inorganic pyrophosphate. The accepted activity name specifies both compatible terminal groups and an energy-coupled reaction.^[ref]

The reaction proceeds through distinct activation states. An adenylate group first attaches to a lysine residue of the RNA ligase, then transfers to the RNA $5^{\prime }$ phosphate. Attack by the $3^{\prime }$ hydroxyl group completes the junction and releases the adenylate as adenosine monophosphate.^[ref] The enzyme can enter another catalytic cycle, whereas the ATP-derived chemical inventory has changed.

For an idealized inventory, start with four nanomoles of one RNA end, three nanomoles of its compatible partner and two nanomoles of ATP. Assume initially unactivated enzyme, no preactivated RNA and no nucleotide regeneration. At most two nanomoles of joined product can form through the stated route, leaving at least two and one nanomoles of the respective fragments. A smaller enzyme amount can still process multiple substrate molecules over repeated cycles; the amount and activity of enzyme affect how quickly that limit is approached.

The original T4-induced ligase purification linked ATP dependence and end modification to measurable products, including evidence for intramolecular circularization under the tested conditions.^[ref] The end requirements distinguish this route from RtcB's GTP-dependent chemistry.

A circular RNA has a covalently closed RNA backbone with no free $5^{\prime }$ or $3^{\prime }$ end. The closure concerns the chain of phosphodiester bonds, not whether a drawing happens to look round. A linear RNA folded into a stem-loop still has two ends. Circular RNAs can arise through RNA ligation; some cellular examples are generated by back-splicing of transcribed sequences.^[ref][ref]

One internal cut in the circle produces a single linear chain. One internal cut in an already linear chain produces two fragments, assuming no other links hold the pieces together. This connectivity difference explains why enzymes requiring a free end can distinguish the molecules, although circularity does not make an RNA immune to every cleavage process.

A 2013 study combined RNA sequencing with and without treatment by RNase R, an enzyme acting from RNA ends. Enrichment of back-splicing junction reads, together with additional validation, supported a substantial population of endogenous circular molecules.^[ref] Combining chemical resistance with junction evidence is more informative than classifying a molecule solely by its apparent shape or persistence after one treatment.

An RNA cyclic phosphate end is an RNA terminus in which a phosphate group links the $2^{\prime }$ and $3^{\prime }$ oxygens of the terminal ribose, forming a small cyclic phosphodiester bond arrangement. The ring is local to the terminal nucleotide; the whole RNA chain need not be circular. This end chemistry differs from both a free $3^{\prime }$ hydroxyl group and an ordinary terminal $3^{\prime }$ phosphate.^[ref]

The difference matters for RNA ligation. A 2012 study showed that RtcB opens the cyclic end to a $3^{\prime }$ phosphate before joining it to an RNA $5^{\prime }$ hydroxyl group. The reactions depended on GTP and enzyme guanylylation. Researchers followed labelled products over time using denaturing polyacrylamide gel electrophoresis, distinguishing starting cyclic ends, transient products and joined RNA.^[ref]

As a measurement example, two four-nucleotide RNA molecules could both spell AUGC while one ends in a free $3^{\prime }$ hydroxyl group and the other in a cyclic phosphate. A readout reporting only their letters would merge them, even though a reaction requiring the free hydroxyl group could distinguish them. Nucleotide sequence therefore does not completely specify terminal chemistry, and terminal chemistry can determine which RNA molecules an assay records.

RNA ligation joins compatible RNA termini by forming a new phosphodiester bond. Joining two fragments extends the connected chain; joining the two ends of one fragment produces circular RNA. The end chemistry matters: an ATP-dependent RNA ligase can join a $3^{\prime }$ hydroxyl group to a $5^{\prime }$ phosphate, whereas RtcB uses a different combination of terminal groups.^[ref][ref]

An unmodified linear four-nucleotide fragment has three backbone links, and a six-nucleotide fragment has five. One intermolecular ligation gives ten nucleotides connected by $3+5+1=9$ links in a linear chain. Circularizing that chain adds one more backbone link while retaining ten nucleotides. Counting chain units, connecting bonds and separate molecules therefore answers three different questions.

A 1972 experiment made end joining measurable using radioactively labelled RNA. After reaction with a purified RNA ligase, labelled terminal phosphate became resistant to alkaline phosphatase. Additional tests with enzymes attacking from chain ends supported formation of circular poly(A) under the studied conditions.^[ref] A single protected label would show a chemical change at that end; the complementary end-accessibility tests made the circular-product interpretation stronger.

RtcB is an RNA ligase that seals an RNA $3^{\prime }$ phosphate to a $5^{\prime }$ hydroxyl group. It also accepts an RNA cyclic phosphate end, which is first opened to a linear $3^{\prime }$ phosphate. Its reaction class, EC6.5.1.8, uses GTP and a divalent metal cofactor; guanosine monophosphate and diphosphate appear in the overall accounting.^[ref]

A 2012 study separated the reaction into experimentally testable steps. GTP first supplies a guanylate group that becomes covalently attached to the enzyme. Transfer to the RNA end creates an activated intermediate; the incoming $5^{\prime }$ hydroxyl group then completes the junction. The authors followed labelled substrates and products using denaturing polyacrylamide gel electrophoresis and found that cyclic-phosphate opening was fast enough to precede joining.^[ref]

A staged interpretation explains a useful experimental distinction. A variant might open a cyclic end yet fail to complete RNA ligation; another defect might prevent the initial enzyme activation. Both could yield little final product, while accumulating different intermediates. Measuring only joined RNA would merge these failures. Resolving the intermediate species helps locate which chemical step has changed.