105
TdT is widely used in molecular biology for applications such as labeling of
3′ends of DNA with modified nucleotides for primer extension DNA sequencing,
generation of DNA blunt ends, in TUNEL (TdT dUTP Nick End Labeling) assay
for detecting apoptosis. Another use of this enzyme is in the addition of complementary homopolymeric tails to linearized plasmid DNA with G’s and the cDNA
with C’s which when incubated together anneal together and can be then transformed into E. coli (Rittié and Perbal 2008).
4.4
DNA-Dependent RNA Polymerases
DNA-dependent RNA polymerases catalyze the transcription process, which is
the 5′ → 3′ synthesis of RNA using a DNA template. Similar to DNA polymerases
lacking exonuclease activity, RNA polymerases are capable of adding an extra
base to the end of a transcript. RNA polymerase was discovered independently by
Charles Loe, Audrey Stevens, and Jerard Hurwitz in 1960. They are ubiquitously
found in all living organisms and also in many viruses. Applications of RNA polymerases in molecular biology include in vitro synthesis of antisense RNA transcripts, labeling of RNA probes, and for RNase protection assay (Melton et al.
1984; Zinn et al. 1983).
DNA-dependent RNA polymerases that are used in in vitro transcription
include E. coli RNA polymerase and bacteriophage RNA polymerases T7, T3,
and SP6 (Struhl 1997). The bacteriophage RNA polymerases are coded by gene 1
present in a family of related bacteriophages. They are capable of catalyzing highyield transcription of DNA sequences present downstream from their promoter,
with tenfold higher elongation rate than that of E. coli RNA polymerase. In addition, their initiation is extremely specific for the individual promoter sequence.
4.4.1 E. coli RNA Polymerase (Holoenzyme)
E. coli RNA polymerase holoenzyme (∼480 kDa) consists of the core enzyme
complex composed of 5 subunits (α 2 ββ′ω) along with sigma factor 70 (σ
70
). RNA
synthesis by this enzyme can be initiated by σ
70
specific bacterial and phage promoters on recognition of the −10 and −35 sequences in the promoter region and
terminate at terminator sequences. Initial studies on in vitro transcription were
carried out using E. coli RNA polymerase (Chamberlin and Berg 1962).
Transcription efficiency is influenced by DNA template quality, promoter strength,
and terminator sequences, in addition to the composition of reaction mixture. E.
coli RNA polymerase holoenzyme can also be used to identify cloned DNA segments containing promoter sequences required for expression in E. coli cells. E.
coli RNA polymerase core enzyme is directly obtained from E. coli cells, while
the sigma factor 70 is obtained after cloning, expression, and purification of the
4 Enzymes as Molecular Tools
TdT is widely used in molecular biology for applications such as labeling of
3′ends of DNA with modified nucleotides for primer extension DNA sequencing,
generation of DNA blunt ends, in TUNEL (TdT dUTP Nick End Labeling) assay
for detecting apoptosis. Another use of this enzyme is in the addition of complementary homopolymeric tails to linearized plasmid DNA with G’s and the cDNA
with C’s which when incubated together anneal together and can be then transformed into E. coli (Rittié and Perbal 2008).
4.4
DNA-Dependent RNA Polymerases
DNA-dependent RNA polymerases catalyze the transcription process, which is
the 5′ → 3′ synthesis of RNA using a DNA template. Similar to DNA polymerases
lacking exonuclease activity, RNA polymerases are capable of adding an extra
base to the end of a transcript. RNA polymerase was discovered independently by
Charles Loe, Audrey Stevens, and Jerard Hurwitz in 1960. They are ubiquitously
found in all living organisms and also in many viruses. Applications of RNA polymerases in molecular biology include in vitro synthesis of antisense RNA transcripts, labeling of RNA probes, and for RNase protection assay (Melton et al.
1984; Zinn et al. 1983).
DNA-dependent RNA polymerases that are used in in vitro transcription
include E. coli RNA polymerase and bacteriophage RNA polymerases T7, T3,
and SP6 (Struhl 1997). The bacteriophage RNA polymerases are coded by gene 1
present in a family of related bacteriophages. They are capable of catalyzing highyield transcription of DNA sequences present downstream from their promoter,
with tenfold higher elongation rate than that of E. coli RNA polymerase. In addition, their initiation is extremely specific for the individual promoter sequence.
4.4.1 E. coli RNA Polymerase (Holoenzyme)
E. coli RNA polymerase holoenzyme (∼480 kDa) consists of the core enzyme
complex composed of 5 subunits (α 2 ββ′ω) along with sigma factor 70 (σ
70
). RNA
synthesis by this enzyme can be initiated by σ
70
specific bacterial and phage promoters on recognition of the −10 and −35 sequences in the promoter region and
terminate at terminator sequences. Initial studies on in vitro transcription were
carried out using E. coli RNA polymerase (Chamberlin and Berg 1962).
Transcription efficiency is influenced by DNA template quality, promoter strength,
and terminator sequences, in addition to the composition of reaction mixture. E.
coli RNA polymerase holoenzyme can also be used to identify cloned DNA segments containing promoter sequences required for expression in E. coli cells. E.
coli RNA polymerase core enzyme is directly obtained from E. coli cells, while
the sigma factor 70 is obtained after cloning, expression, and purification of the
4 Enzymes as Molecular Tools