TEMPLATE REQUIREMENTS

• RNA Polymerases has an absolute requirement

for template satisfied either by native DNA or
heat denatured DNA.
• Calf Thymus DNA was denatured by heating at
95 ̊ C – 98 ̊ C for 10 minutes and followed by
quick cooling. Reactions conditions were carried
out in standard buffer along with primers. Each
reaction mixture contained 0.065M Ammonium
Sulphate.
• At a fixed DNA concentration, the soluble
polymerase activity is proportional to enzyme
concentration. (Jacob et al, 1968).
• Denatured DNA saturates sea-urchin RNA
activities I and II at lower concentrations than
does native DNA.
• While, the saturation curves of form III with the
two DNA templates are similar.
• The relative activity with native and denatured
DNA templates of forms I and II is a function of
ionic strength.
• Experiments with polymerase II show that the
activity with denatured DNA is less sensitive to
the change in ionic strength than in the activity of
the native DNA.
Relative transcription of native and heat-denatured DNA
by sea urchin RNA Polymerases. The amount of enzyme
protein per reaction were 30, 7.4,8.4 μg for forms I, II,
III, respectively.
 TEMPLATE REQUIREMENTS

• At saturating levels of template and at 0.04 M Ammonium Sulphate, the

denatured DNA / native DNA activity ratios of the rat RNA polymerase I
and II are 1:3 and 3:1 respectively.
• Crude-soluble rat liver enzymes show a high denatured DNA/ native DNA
activity ratio and that ratio is a function of salt concentration. This
difference in the activity may be due to the use of calf thymus DNA as the
primer. (Jacob et al, 1968).
• Purified E.coli RNA polymerase can synthesize more RNA molecules on
denatured T4 DNA templates than on the native template. It is possible that
strand separation due to denaturation makes available to the polymerase a
larger number of attachment sites. (Bremer and Bruner, 1968)
INCORPORATION OF RIBONUCLEOSIDE
TRIPHOSPHATES
• Independent incubations were conducted with each labelled nucleotide in the assay
conditions as previously mentioned, except that all nucleotide concentrations were
0.4 mM. The ammonium sulphate concentrations were 0.04 M, 0.08 M, 0.09M for
polymerse I, II and III, respectively.
• The total nucleotide incorporations were 645,819,126, 3668 picomoles/10 min for
polymerase I, II, III and the E.coli enzymes, respectively.
• Significant homopolymer was only detected with ATP.
• The base ratios of the rNA transcribed from native DNA by the sea urchin activities
are similar to each other as well as to those of the RNA produced by the E.Coli
enzyme with the same template.
• From this study, there was no significant indication that the polymerases isolates are
endowed with the capability to transcribe selected regions of a DNA molecule.

Table : Relative nucleotide incorporation by sea urchin RNA Polymerases