Eukaryotic RNA Polymerases 63
cally interacts with RNA polymerase II and TFIIB (26,27). These
transcription factors range in mol mass from 25,000 Da for yeast (28)
to 76,000 Da for TFIIE (24).
Although promoters themselves are sufficient to direct basal level
gene expression, specific transcription requires the assistance of addi-
tional transcription factors (29). These sequence-specific DNA bind-
ing proteins are directly involved in the recognition of mRNA
transcription initiation in higher organisms; for example, CAT tran-
scription factor (CTF) is responsible for selective recognition of eukary-
otic promoters that contain the sequence CCAAT (30,31), whereas the
Sp 1 protein binding regions contain one or more GC boxes (GGGCGG),
which can be present in either orientation with respect to transcription.
Not all GC boxes bind Sp 1 with equal affinity, and sequences outside
the core hexanucleotide seem to modulate the efficiency of binding
(for review, see refs. 25,32).
In HeLa cell factor, termed the major late transcription factor or
upstream stimulatory factor, MLTF/USF binds to an upstream consen-
sus sequence GGCCACGTGACC, which functions in a bidirectional
manner. MLTF/USF may act synergistically with TFIID, since binding of
MLTF/USF enhances binding of TFIID (33, 34). The binding sequence
occurs in a number of cellular promoters, for example, mouse and rat
y-fibrinogen (35). Another promoter-specific transcription factor is
the heat shock transcription factor (HSTF). All maj or heat shock genes
contain multiple HSE (heat shock regulatory elements) with the consen-
sus sequence C--GAA--TCC-----G (36). In HeLacells, heat shock causes
more HSTF to bind to the HSE, whereas in yeast, HSTF binds consti-
tutively and heat shock causes HSTF to become phosphorylated (37).
3.1. In Vitro Transcription Systems
The development of methods for preparing soluble whole-cell and
nuclear extracts from mammalian cells (38-41) has led to a dramatic
increase in our understanding of the mechanistic details of complex
biochemical reactions, such as transcription, replication, and RNA
processing.
In vitro transcription systems were partially developed to study the
biochemical mechanisms of DNA-protein interaction, gene expres-
sion, and regulation. To date, several cell-free transcription systems
have been developed, the first by Wu in 1978 (42); which demon-