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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.

An RNA ligase is an enzyme that catalyzes covalent joining of RNA ends. Its usefulness depends on the terminal groups it recognizes and the chemistry that activates the joining reaction. The activity classified as EC6.5.1.3, an ATP-dependent RNA ligase, joins $3^{\prime }$ hydroxyl group and $5^{\prime }$ phosphate termini while converting ATP to adenosine monophosphate and diphosphate.^[ref]

RtcB illustrates another route. It joins a $3^{\prime }$ phosphate to a $5^{\prime }$ hydroxyl group using GTP, and can process an RNA cyclic phosphate end before sealing. The substrate ends appropriate for one RNA ligase therefore need not be appropriate for another.^[ref]

For a specified assay, imagine two RNA fragments held in the correct alignment. One pair has the terminal groups accepted by the chosen RNA ligase; an otherwise sequence-identical pair carries a blocked end. A failure to join the second pair could reflect end chemistry even though the letters and alignment match. A meaningful comparison separates recognition, activation and final phosphodiester bond formation. Historically, purification of a T4-induced enzyme and tests of its labelled products connected the joining activity to a biochemical preparation.^[ref] That functional evidence goes beyond assigning an enzyme name from sequence resemblance alone.

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.