64 Cook and Slaterstrated accurate transcription of exogenous DNA by polymerase III,
and others such as those developed by Weil et al. (38); Manley et al.
(39); Dignam et al. (40); and Zerivitz and Akusjarvi (41).
The major advantage of soluble cell-free transcription systems is
that they can be fractionated by standard techniques in order to iden-
tify those components involved in the transcription process (43). The
purified enzymes alone do not direct selective and accurate transcription
from purified DNA templates, but they appear to initiate and terminate
at a large number of sites, which may not be relevant to transcription
in vivo (3,4). The most extensive use of in vitro transcription systems
to date has been to study the structure of promoter sites and the tran-
scription factors needed to bring about selective transcription (44).
- Experimental Procedures
4.1. TCA Precipitation Assay
This procedure can be used as an estimate of nucleoside triphos-
phate incorporation into trichloroacetic acid (TCA) precipitable prod-
ucts following an in vitro transcription reaction. 3H-UTP is usually the
radiolabel of choice. The reaction (as detailed in Section 4.2., but for
the substitution of 32p-labeled GTP with 1.0 ~tCi of 3H-UTP) is stopped
by the addition of 10% TCA. Acid-insoluble material is then collected
on glass fiber disks, washed twice with 5% TCA (with sodium pyro-
phosphate to reduce nonspecific binding of the radiolabel), and finally
with alcohol to remove unincorporated radiolabel. The filters are dried
and the radioactivity estimated by using a scintillation counter; hence,
an estimation of nucleotide incorporation can be made (1).
4.2. Run-Off Transcription
RNA polymerase does not accurately terminate transcription in
vitro. One procedure therefore is to use as template a cloned gene
fragment that includes the promoter, but is truncated downstream.
Using the "run-off" assay system, the whole-cell or nuclear lysate,
DNA template, and radiolabeled (32p) nucleotide are incubated at 30°C
for 60 min. RNA is synthesized from the truncated DNA template and
analyzed electrophoretically to detect the species of RNA with the
predicted length.
A typical 50-]xL in vitro transcription reaction would contain the
following: