224 Maunders4.2. Enzymic Reaction
4.2.1. Nucleic Acid Substrate
The normal substrate ofRNA ligase is single-stranded RNA, although
it will act on a variety of single- or double-stranded RNA or DNA mol-
ecules (60-68). RNA ligase will act on very small pieces of ribonucleic
acid, with 40 mers being the probable upper size limit (69). The minimum
size of the 5' moiety is a ribonucleoside 3'5'-bis phosphate (i.e., P-base-P),
a 5'-monophosphate being ineffective. The nature of the base has some
bearing on the reaction rate, pyrimidines reacting 2-10 times faster than
purines, and modified purines slower yet. With these reservations, a wide
variety of bases will be accepted, including deoxynucleosides, O-methy-
lated-, halogenated-, dihydro-, thio-, and deaza-derivatives, and deami-
nated purines (58). The minimum size of the 3' moiety is a trinucleoside
bis-phosphate, base-P-base-P-base-OH.
4.2.2. Cofactors
The enzyme requires ATP (although dATP will substitute) and forms
an enzyme-AMP complex during reaction. Low levels of ATP are
preferred, often provided by an ATP-generating system (58).
4.2.3. Cations
Magnesium ions are required for reaction, although Mn 2÷ will sub-
stitute and often improve the rate of reaction; 1M (NH4)2SO 4 com-
pletely inhibits activity.
4.2.4. Enzyme Assay
The enzyme is usually assayed by measuring the conversion of 5'-
32p-labeled poly-A to a circular form insensitive to phosphomo-
noesterase at 37°C. One unit converts 1 nmol of 5'-32p-labeled poly-A
at a concentration of 1 ~/(24) or 10 l.lM (59) termini to a phosphatase-
resistant form in 30 min at 37°C.
Another definition of the unit of activity is that it ligates 1 pmol of
32p-labeled mononucleoside biphosphate to a larger TCA-precipitable
RNA species in 30 min at 4°C. This unit is approximately equivalent
to one-sixth of that defined by the phosphatase resistance assay.
A third unit can be defined on the basis of the pyrophosphate exchange
reaction. One unit of enzyme catalyzes the exchange of 1 nmol of 32p_
labeled inorganic pyrophosphate into ATP in 30 min.