108
(Makeyev and Bamford 2000). The phi6 RNA polymerase can generate labeled
RNA that can be used as a probe in hybridization and in the synthesis of dsRNA
used in RNA interference (RNAi) for in vivo gene silencing. Another application is
in primer-independent RNA sequencing using chain terminators, (RdRP sequencing) where 3′ proximal or even complete sequence of target RNA molecules can be
rapidly deduced without prior sequence information. This has applications in RNA
virus research and diagnostics (Makeyev and Bamford 2001).
4.6
Template-Independent RNA Polymerases
Majority of the RNA molecules in cells are synthesized from a DNA template. In
some cases, like poly(A) tailing of messenger RNA, RNA synthesis occurs without
a template.
4.6.1 Poly(A) Polymerase
It is a template-independent polymerase that synthesizes the poly(A) (adenine) tail
at the 3′ end of messenger RNA. By using ATP as precursor, poly(A) polymerase
catalyzes the addition of AMP residues to the free 3′-hydroxyl terminus of RNA
(Edmonds 1982). It highly prefers ATP when compared to other NTPs. Varying
lengths of poly(A) tail can be synthesized by adjusting the time of incubation,
enzyme quantity, and available number of 3′ termini for addition. Poly(A) polymerase from E. coli and yeast are used in molecular biology for RNA tailing and 3′
end labeling. Poly(A)-tailed RNA molecules can be PCR amplified using oligo(dT)
primers by reverse transcription, and the resultant cDNA can be used in cloning.
Since poly(A) polymerase can incorporate modified nucleotides like biotin-N6-ATP
and digoxigenin-11-UTP with high efficiency, it can be used to synthesize RNA
molecules with radioactive or fluorescent label for using as probes in various applications (Martin and Keller 1998).
4.7
Reverse Transcriptases
They are RNA-dependent DNA polymerases that catalyze the synthesis of a DNA
molecule complementary to a given RNA template. Reverse transcriptases are
encoded by retroviruses and are essential for their life cycle. The viral RNA genome
is converted into DNA via reverse transcriptase and the synthesized proviral dsDNA
molecule integrates into host cells during infection. Under in vitro conditions,
reverse transcriptase can synthesize DNA using either ssRNA or ssDNA templates
with the help of a primer. Reverse transcriptases have lower fidelity than DNAdependent DNA polymerases since they lack 3′ → 5′ exonuclease proofreading
activity.
G. Valsala and S. Sugathan
(Makeyev and Bamford 2000). The phi6 RNA polymerase can generate labeled
RNA that can be used as a probe in hybridization and in the synthesis of dsRNA
used in RNA interference (RNAi) for in vivo gene silencing. Another application is
in primer-independent RNA sequencing using chain terminators, (RdRP sequencing) where 3′ proximal or even complete sequence of target RNA molecules can be
rapidly deduced without prior sequence information. This has applications in RNA
virus research and diagnostics (Makeyev and Bamford 2001).
4.6
Template-Independent RNA Polymerases
Majority of the RNA molecules in cells are synthesized from a DNA template. In
some cases, like poly(A) tailing of messenger RNA, RNA synthesis occurs without
a template.
4.6.1 Poly(A) Polymerase
It is a template-independent polymerase that synthesizes the poly(A) (adenine) tail
at the 3′ end of messenger RNA. By using ATP as precursor, poly(A) polymerase
catalyzes the addition of AMP residues to the free 3′-hydroxyl terminus of RNA
(Edmonds 1982). It highly prefers ATP when compared to other NTPs. Varying
lengths of poly(A) tail can be synthesized by adjusting the time of incubation,
enzyme quantity, and available number of 3′ termini for addition. Poly(A) polymerase from E. coli and yeast are used in molecular biology for RNA tailing and 3′
end labeling. Poly(A)-tailed RNA molecules can be PCR amplified using oligo(dT)
primers by reverse transcription, and the resultant cDNA can be used in cloning.
Since poly(A) polymerase can incorporate modified nucleotides like biotin-N6-ATP
and digoxigenin-11-UTP with high efficiency, it can be used to synthesize RNA
molecules with radioactive or fluorescent label for using as probes in various applications (Martin and Keller 1998).
4.7
Reverse Transcriptases
They are RNA-dependent DNA polymerases that catalyze the synthesis of a DNA
molecule complementary to a given RNA template. Reverse transcriptases are
encoded by retroviruses and are essential for their life cycle. The viral RNA genome
is converted into DNA via reverse transcriptase and the synthesized proviral dsDNA
molecule integrates into host cells during infection. Under in vitro conditions,
reverse transcriptase can synthesize DNA using either ssRNA or ssDNA templates
with the help of a primer. Reverse transcriptases have lower fidelity than DNAdependent DNA polymerases since they lack 3′ → 5′ exonuclease proofreading
activity.
G. Valsala and S. Sugathan