Download Promega Notes: T4 RNA Ligase: A Molecular Tool for RNA and DNA

Promega Notes Number 65, 1998, p. 07
T4 RNA Ligase: A Molecular Tool for RNA and DNA
Manipulations
By Ken Lewis, Fen Huang and Greg Beckler
Promega Corporation
Corresponding author: e-mail to [email protected]
T4 RNA ligase catalyzes the ligation of two strands of RNA between the 5´-phosphate and 3´-hydroxyl groups. The newly introduced T4
RNA Ligase from Promega is useful for this application and labeling RNA at the 3´-end.
INTRODUCTION
T4 RNA ligase is useful for joining RNA to RNA when the donor molecule contains a 5´-phosphate group (PO4) and the acceptor
molecule contains a 3´-hydroxyl group (OH). Circularization of RNA molecules is also possible. Like many of the bacteriophage T4encoded enzymes, the function of T4 RNA ligase in the life cycle of the bacteriophage is speculative. RNA ligase was discovered by
Hurwitz and colleagues at the Albert Einstein College of Medicine in 1972 (1-3). Even though other T-even bacteriophages, but not
lambda or T-odd bacteriophages, produce RNA ligase, only the enzyme from bacteriophage T4 has been purified and studied in any
detail (2).
T4 RNA ligase has been cloned and overexpressed at Promega. The protein is 43.5kDa, acidic and binds tightly to DEAE resins.
Promega's T4 RNA Ligase (Cat.# M1051) has been purified to physical homogeneity and is cleaner than RNA ligases available from
competing vendors (see Figure 1).
Figure 1. Purity of Promega's T4 RNA Ligase and three other commercially available enzymes. Twenty
units of RNA ligase from Vendors A, B, C and Promega were resolved on a 4-20% NOVEXTM
polyacrylamide gel containing SDS and stained with Coomassie® brilliant blue.
ACTIVITIES AND USES OF T4 RNA LIGASE
T4 RNA ligase is predominantly useful for joining RNA to RNA. As stated above, both a 5´-PO4-bearing donor and a 3´-OH-bearing
acceptor are required. DNA may also serve as a donor, but is a poor acceptor (4). T4 RNA ligase can join DNA to DNA, mainly in an
intramolecular reaction, but with very low efficiency. This property has been used in the cDNA circularization reaction of the 5´ RACE
or Rapid Amplification of cDNA Ends technique of Maruyama et al. (5).
Joining short DNA oligos to each other using T4 RNA ligase can be difficult. McCoy and Gumport (6) have reported that this reaction
requires manganese, low levels of ATP, an ATP-regenerating system and high concentrations of the RNA ligase. However, we routinely
ligate short RNA oligonucleotides onto the 5´-end of decapped eukaryotic messenger RNA. The short RNA oligonucleotide serves as an
anchor in the 5´-RACE technique of Schaefer (7) where the goal is capture of the complete 5´-end sequence information of mRNA by
PCR*. The RNA tag that is attached at the 5´-end of the message serves, following reverse transcription, as a PCR priming site that
enables amplification of the 5´-end of the mRNA.
*The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of
the PCR process.
At Promega, we assay T4 RNA Ligase by converting 5´-32P-labeled poly(A)14-20 to a phosphatase-resistant, circular form of the
molecule. In studies of the circularization reaction of poly(A) n, it was found that the minimum size of poly(A)n addition for the reaction
to proceed was n=8 and that the reaction peaked at n=10-16 (8). The reaction then decreased as the chain length increased up to n=100
(8). One unit is defined as the amount of enzyme that catalyzes transformation of one nanomole of 5´-[32 P]poly(A) 14-20 into a
phosphatase-resistant form in 30 minutes at 37°C at a 10µM concentration of 5´-ends.
The ligation reaction requires ATP. Following adenylation of the enzyme by covalent attachment of AMP, the AMP is transferred to the
donor and the 3´-OH group of the acceptor attacks this activated bond to yield a phosphodiester bond between the donor and acceptor
molecules (2).
RNA ligase has been used to specifically label the 3´-end of RNA (9). In this reaction, 32P is incorporated as [32P]pCp where the
mononucleotide is the donor and the RNA molecule is the acceptor.
BUILDING SHORT RNAs
England and Uhlenbeck have discussed the use of T4 RNA ligase for the enzymatic synthesis of oligoribonucleotides of defined
sequence (10). These investigators examined the joining of a number of donors and acceptors of different lengths and sequences. They
found that the smallest possible donor was a nucleoside 3´,5´-biphosphate. The 3´-PO4 group was required as 5´-AMP or pA2 ´p did not
function. For longer donors, the 3´-PO4 group is not necessary; however, the 3´-PO4 group prevents the intramolecular circularization
of the donor or intermolecular donor-donor dimerization and thus ensures the synthesis of a unique product. Following ligation, the 3´PO4 group may be removed by alkaline phosphatase and the product used as the acceptor in another reaction. The minimum acceptor
length was found to be a trinucleotide, with little increase in reaction rate as chain length increased (10). The base composition of the
acceptor was found to markedly influence the reaction rate while the composition of the donor had little effect. Chemical methods to
synthesize oligoribonucleotides are expensive and are not useful for longer sequences. The enzymatic joining of shorter, synthetic
oligoribonucleotides with T4 RNA Ligase may be a convenient method for preparing longer RNAs of defined sequence.
PROTEIN LABELING
In 1989, Peter Schultz and colleagues at the University of California-Berkeley improved a general method for site-specific incorporation
of unnatural amino acids into proteins (11,12). The improvement consisted of using T4 RNA ligase to enzymatically join a run-off
amber suppressor tRNA lacking the terminal 3´-CA sequence to a CA dinucleotide that had been chemically modified with an unnatural
amino acid. This improvement greatly simplified the original anticodon loop replacement procedure, and they demonstrated that, while
lacking post-transcriptional base modifications, the efficiency of amber suppression of the run-off suppressor tRNA was equivalent to a
suppressor tRNA constructed by anticodon replacement (12). This approach has been used to successfully incorporate a variety of
probes in a site-specific manner. Examples include incorporation of spin-labels, fluorescent and photoactivatable amino acids into
proteins such as T4 lysozyme or nicotinic acetylcholine receptor (13,14).
SUMMARY
Promega's T4 RNA Ligase is >90% homogeneous. It is ideal for joining RNA to RNA, in single-stranded form, in an intermolecular
reaction and for circularizing ssRNA in an intramolecular reaction. Using the right conditions and correct phosphate and hydroxyl
groups, T4 RNA Ligase can be used to add ribonucleotides to mRNA.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Leis, J. et al. (1972) In: Advances in the Biosciences, Raspe, G., ed., Vol. VIII, Permagon Press, New York, 117.
Uhlenbeck, O.C. and Gumport, R.I. (1982) In: The Enzymes, Boyer, P.D., ed., Vol. XV, Part B, 31.
Silber, R., Malathi, V.G. and Hurwitz, J. (1972) Proc. Natl. Acad. Sci. USA 69, 3009.
Brennan, C.A., Manthey, A.E. and Gumport, R.I. (1983) Meth. Enzymol. 100, 38.
Maruyama, I.N., Rakow, T.L. and Maruyama, H.I. (1995) Nucl. Acids Res. 23, 3796.
McCoy, M.I.M. and Gumport, R.I. (1980) Biochemistry 19, 635.
Schaefer, B.C. (1995) Anal. Biochem. 227, 255.
Kaufmann, G., Klein, T. and Littauer, U.Z. (1974) FEBS Lett. 46, 271.
England, T.E., Bruce, A.G. and Uhlenbeck, O.C. (1980) Meth. Enzymol. 65, 65.
England, T.E. and Uhlenbeck, O.C. (1978) Biochemistry 17, 2069.
Noren, C.J. et al. (1989) Science 244, 182.
12. Noren, C.J. et al. (1990) Nucl. Acids Res. 18, 83.
13. Cornish, V.W. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 2910.
14. Nowak, M.W. et al. (1995) Science 268, 439.
Ordering Information
Product
Size
Cat.#
T4 RNA Ligase
500u
M1051
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