107
4.4.4 SP6 RNA Polymerase
SP6 RNA polymerase was isolated from SP6 bacteriophage-infected Salmonella
typhimurium LT2. It is of 96 kDa size and requires Mg
2+
for its activity. This enzyme
has been found to be activated by spermidine and serum albumine (Butler and
Chamberlin 1982). SP6 shows high promoter specificity and can only transcribe
DNA segments inserted downstream to their specific promoter. It also has the ability
to transcribe through poly(A) stretches (Melton et al. 1984). SP6 RNA polymerase
can be used for all techniques mentioned for T7 RNA polymerase. SP6 polymerase
requires a complete double-stranded DNA template for its activity, in contrast to T7
RNA polymerase, which has high efficiency when 18 base promoter regions are
double-stranded. RNA yield is much higher for SP6 polymerase in comparison to
T7 polymerase, and on scale up of the reaction, even milligram quantities of RNA
can be obtained.
4.4.5 Thermus RNA Polymerase
Thermus RNA polymerase is isolated from Thermus aquaticus, a thermophile, and
is the only commercially available RNA polymerase that can remain stable and
function at temperatures higher than 65 °C. It is structurally similar to E. coli RNA
polymerase and has the same promoter requirements. However, Thermus RNA
polymerase is resistant to several inhibitors of the E. coli enzyme, including fidaxomicin and rifampin. It has low transcription termination efficiently and has high
rate of intrinsic transcript cleavage. Commercial production of this enzyme is by
cloning and over expression of Thermus RNA polymerase gene in E. coli (Minakhin
et al. 2001).
4.5
RNA-Dependent RNA Polymerases
RNA-dependent RNA polymerases (RdRp) are enzymes that catalyze RNA strand
synthesis complementary to a given RNA template. They are encoded by all viruses
with negative sense RNA as its genetic material. These enzymes transcribe the RNA
into messenger RNA with the help of viral and host factors. A number of eukaryotes
have also been found to produce putative RdRps that are considered to be involved
in RNA-triggered sequence-specific gene silencing.
4.5.1 phi6 RNA Polymerase
An RNA-dependent RNA polymerase from Pseudomonas bacteriophage phi6, it
has high processivity and ability to act on single- or double-stranded RNA template.
Even though it does not require a primer for initiation, the sequence
5′-NNUUUUUUUUCC-3′ is essential on the template strand for optimal initiation
4 Enzymes as Molecular Tools
4.4.4 SP6 RNA Polymerase
SP6 RNA polymerase was isolated from SP6 bacteriophage-infected Salmonella
typhimurium LT2. It is of 96 kDa size and requires Mg
2+
for its activity. This enzyme
has been found to be activated by spermidine and serum albumine (Butler and
Chamberlin 1982). SP6 shows high promoter specificity and can only transcribe
DNA segments inserted downstream to their specific promoter. It also has the ability
to transcribe through poly(A) stretches (Melton et al. 1984). SP6 RNA polymerase
can be used for all techniques mentioned for T7 RNA polymerase. SP6 polymerase
requires a complete double-stranded DNA template for its activity, in contrast to T7
RNA polymerase, which has high efficiency when 18 base promoter regions are
double-stranded. RNA yield is much higher for SP6 polymerase in comparison to
T7 polymerase, and on scale up of the reaction, even milligram quantities of RNA
can be obtained.
4.4.5 Thermus RNA Polymerase
Thermus RNA polymerase is isolated from Thermus aquaticus, a thermophile, and
is the only commercially available RNA polymerase that can remain stable and
function at temperatures higher than 65 °C. It is structurally similar to E. coli RNA
polymerase and has the same promoter requirements. However, Thermus RNA
polymerase is resistant to several inhibitors of the E. coli enzyme, including fidaxomicin and rifampin. It has low transcription termination efficiently and has high
rate of intrinsic transcript cleavage. Commercial production of this enzyme is by
cloning and over expression of Thermus RNA polymerase gene in E. coli (Minakhin
et al. 2001).
4.5
RNA-Dependent RNA Polymerases
RNA-dependent RNA polymerases (RdRp) are enzymes that catalyze RNA strand
synthesis complementary to a given RNA template. They are encoded by all viruses
with negative sense RNA as its genetic material. These enzymes transcribe the RNA
into messenger RNA with the help of viral and host factors. A number of eukaryotes
have also been found to produce putative RdRps that are considered to be involved
in RNA-triggered sequence-specific gene silencing.
4.5.1 phi6 RNA Polymerase
An RNA-dependent RNA polymerase from Pseudomonas bacteriophage phi6, it
has high processivity and ability to act on single- or double-stranded RNA template.
Even though it does not require a primer for initiation, the sequence
5′-NNUUUUUUUUCC-3′ is essential on the template strand for optimal initiation
4 Enzymes as Molecular Tools