62 Cook and Slater
2.2. Ion Requirement
The requirement for a divalent cation by RNA polymerases indicates
binding of nucleotides to the enzyme as metal chelates, for example:
MgATP (11). The optimum divalent cation concentration is generally
in the range of 5-10 mM Mg 2÷ or 1-2 mM Mn 2÷ (2). In addition to the
above requirements, RNA polymerase activity is stimulated by the
inclusion of monovalent cations, such as (NH4)2SO 4, NH4C1, KC1, or
NaC1. This is especially evident when engaged RNA polymerases are
assayed in isolated nuclei or chromatin. With isolated plant or animal
nuclei, RNA polymerase I and III activity is most active at 0.05-0.10M
(NH4)2SO4, whereas RNA polymerase II activity is optimized at 0.25-
0.50M (12-15).
2.3. Structure
The nuclear RNA polymerases have a complex subunit structure.
Each has a mol mass of approx 500,000 Da and is made up of two large
subunits, each having a mol mass >100,000 Da, which seem to be
characteristic for each class of enzyme, and a number of smaller sub-
units, with mol wt from 10,000 Da to just less than 100,000 Da (2,4).
The situation is further complicated by the phenomenon of
microheterogeneity within RNA polymerase classes (7).
- Transcription Factors
The basic components required for efficient and accurately regu-
lated eukaryotic transcription initiation include two types of DNA
elements known as promoters and upstream regulatory sequences;
two sets of proteins known as general transcription factors and spe-
cific or regulatory proteins, and the RNA polymerase enzyme. Tran-
scription can occur when the eukaryotic RNA polymerase recognizes
a preexisting DNA-protein complex. RNA polymerase II has four
general transcription factors associated with it; these have been par-
tially purified and designated TFIIA, B, D, and E (16-19). TFIIA is
required for the efficient interaction of TFIID with the promoter ele-
ment known as the TATA box (20,21), which is found in nearly all
eukaryotic promoters, with a consensus sequence "TATAAA" and
located approx 25-30 bp upstream of the transcription start site in
mammalian promoters (22-25). It has been shown that TFIIE specifi-
