Methods in Molecular Biology • 16 Enzymes of Molecular Biology

Eukaryotic RNA Polymerases

Table 1

Properties of Eukaryotic RNA Polymerases a

61

Eukaryotic RNA polymerases

I II III

Cellular location

Transcription product

~-Amanitin sensitivity

(lag/mL required for

50% inhibition)

Subunit structure

Ionic strength optima

(NH4)2SO 4 M

Soluble enzyme

Chromatin-bound
enzyme
Mn2+/Mg 2+ activity ratio
Poly (dA/dT)/DNA activity
ratio
Chromatographic elution
(NH4)2SO 4 M
DEAE-Sephadex
DEAE-cellulose

Nucleolus Nucleoplasm Nucleoplasm

rRNA hnRNA tRNA

snRNA 5SrRNA

Nonsensitive Very sensitive Moderately

(0.01-0.05) sensitive

(5-1000)

Two large subunits 100,000-240,000

and several small subunits 10,000-95,000

0.03-0.05 0.025--0.1 0.04-0.01,

0.18-0.3

0.05-0.1 0.25-0.5 0.05-0.1

~5:1 -7:1 ~3:1

2:3 1:2 1:3

~0.1 -0.2 0.2-0.3

-0.1 -0.2 -0.1

~I'aken from refs. 1,4,6,7.

extracts, it is apparent that additional factors are required to obtain faithful
transcription in vitro, so the enzymes are not often used in their pure state
because they lack specificity. Eukaryotic RNApolymerases differ in subunit
structure, type of genome transcribed, chromatographic properties, diva-
lent cation, ionic strength optima, and ct-amanitin sensitivity. The poison-
ous mushroom Amanita phalloides produces ct-amanitin, a bicyclic
octopeptide that blocks the elongation step of transcription by direct
interaction with RNApolymerases that differ in their sensitivity (Table 1).
RNA polymerase I is insensitive to the toxin, but is responsive to actino-
mycin D, which binds to DNA preventing elongation. RNA polymerase
II is very sensitive and RNA polymerase III moderately so. Thus, the
various RNApolymerases I, II, and III are readily distinguished (Table 1).
Intact cells are not freely permeable to ot-amanitin; therefore, it is neces-
sary to use a whole-cell or nuclear extract.