106
protein in E. coli. Bacteriophage RNA polymerases have replaced this enzyme for
most applications due to the following demerits: detachment of subunits leading to
premature termination of in vitro and incapability in synthesizing uniform RNA
population (Chamberlin and Ryan 1982). E. coli RNA polymerase holoenzyme is
still in use for synthesizing transcripts for cloning into vectors lacking phage RNA
polymerase promoter. The core enzyme devoid of the σ subunit can synthesize uniform short transcripts from DNA template under conditions of high concentrations
of random primers and low concentrations of NTP (nucleoside triphosphate).
4.4.2 T7 RNA Polymerase
T7 RNA polymerase is a polypeptide of 98 kDa size isolated from T7 bacteriophage
(Stahl and Zinn 1981). It has high promoter specificity and can carry out transcription of DNA present downstream from a T7 promoter. RNA synthesis by this
enzyme requires a double-stranded DNA template in addition to Mg
2+
as cofactor
(Chamberlin et al. 1970). T7 RNA polymerase is usually stimulated by BSA or
spermidine. It has a very low error rate and can transcribe through poly(A) regions,
which helps in multiple transcriptions of circular DNA template without dissociation. T7 RNA polymerase is usually employed in the transcription of DNA cloned
into vectors containing two different phage promoters in opposite orientations.
RNA synthesis can be carried out selectively from either of the inserted DNA
strands using different polymerases. Commercial production is from E. coli cloned
and expressed with T7 gene I. The major applications of this enzyme include generation of homogeneously labeled single-stranded RNA, nonisotopic labeling, RNA
vaccine synthesis and production of antisense RNA for gene expression
experiments.
4.4.3 T3 RNA Polymerase
E. coli bacteriophage T3 RNA polymerase is a 99 kDa enzyme that catalyzes in
vitro RNA synthesis from a cloned DNA sequence under the T3 promoter. It is
coded by gene I of T3 phage. The amino acid composition of this polymerase shows
82% identity to T7 RNA polymerase. Promoter sequence for the T3 RNA polymerase is present 12 base pairs away from the stop codon of structural gene. This
enzyme can be used to generate huge quantities of particular RNA transcripts from
cloned vectors containing T3 promoter. Such RNA transcripts have application in
Northern and Southern blotting as probes, studies on in vitro translation and RNA
processing, manufacture of RNA vaccines, and for exon-intron mapping of genomic
DNA (McGraw et al. 1985). It can polymerize both radioactively labeled and
hapten- labeled nucleoside triphosphates.
G. Valsala and S. Sugathan
protein in E. coli. Bacteriophage RNA polymerases have replaced this enzyme for
most applications due to the following demerits: detachment of subunits leading to
premature termination of in vitro and incapability in synthesizing uniform RNA
population (Chamberlin and Ryan 1982). E. coli RNA polymerase holoenzyme is
still in use for synthesizing transcripts for cloning into vectors lacking phage RNA
polymerase promoter. The core enzyme devoid of the σ subunit can synthesize uniform short transcripts from DNA template under conditions of high concentrations
of random primers and low concentrations of NTP (nucleoside triphosphate).
4.4.2 T7 RNA Polymerase
T7 RNA polymerase is a polypeptide of 98 kDa size isolated from T7 bacteriophage
(Stahl and Zinn 1981). It has high promoter specificity and can carry out transcription of DNA present downstream from a T7 promoter. RNA synthesis by this
enzyme requires a double-stranded DNA template in addition to Mg
2+
as cofactor
(Chamberlin et al. 1970). T7 RNA polymerase is usually stimulated by BSA or
spermidine. It has a very low error rate and can transcribe through poly(A) regions,
which helps in multiple transcriptions of circular DNA template without dissociation. T7 RNA polymerase is usually employed in the transcription of DNA cloned
into vectors containing two different phage promoters in opposite orientations.
RNA synthesis can be carried out selectively from either of the inserted DNA
strands using different polymerases. Commercial production is from E. coli cloned
and expressed with T7 gene I. The major applications of this enzyme include generation of homogeneously labeled single-stranded RNA, nonisotopic labeling, RNA
vaccine synthesis and production of antisense RNA for gene expression
experiments.
4.4.3 T3 RNA Polymerase
E. coli bacteriophage T3 RNA polymerase is a 99 kDa enzyme that catalyzes in
vitro RNA synthesis from a cloned DNA sequence under the T3 promoter. It is
coded by gene I of T3 phage. The amino acid composition of this polymerase shows
82% identity to T7 RNA polymerase. Promoter sequence for the T3 RNA polymerase is present 12 base pairs away from the stop codon of structural gene. This
enzyme can be used to generate huge quantities of particular RNA transcripts from
cloned vectors containing T3 promoter. Such RNA transcripts have application in
Northern and Southern blotting as probes, studies on in vitro translation and RNA
processing, manufacture of RNA vaccines, and for exon-intron mapping of genomic
DNA (McGraw et al. 1985). It can polymerize both radioactively labeled and
hapten- labeled nucleoside triphosphates.
G. Valsala and S. Sugathan