MOLECULAR ANALYSIS OF RNA POLYMERASE II 
JEFFRY L. CORDEN, PH.D., Assoctate Investigator 
Dr. Corden's laboratory has continued to study 
RNA polymerase structure and function. During the 
past year attention has been focused on the un- 
usual repeated sequence at the carboxyl terminus 
of the largest subunit of mouse RNA polymerase II 
(RPII). This domain consists of 52 repeats of a 
seven-amino acid sequence with the consensus se- 
quence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A similar car- 
boxyl-terminal domain (CTD) is found in the larg- 
est subunit of RPII from a variety of species, but it is 
not present in eukaryotic RNA polymerases I or III 
or in prokaryotic RNA polymerases, indicating that 
it plays a unique role in RPII transcription. Ongoing 
studies in Dr. Corden's laboratory are designed to 
elucidate the function of this domain. 
One characteristic of the CTD is a high content of 
amino acids that can be modified by phosphoryla- 
tion. Actively transcribing RPII is highly phosphory- 
lated; analysis of breakdov^^n products of the largest 
subunit suggested that the CTD was the primary 
site of phosphorylation. One objective of Dr. 
Corden's laboratory has been to identify and ana- 
lyze the protein kinase that phosphorylates the 
CTD. The first step was to show that short peptides 
containing several of the seven-amino acid consen- 
sus blocks were phosphorylated in whole-cell ex- 
tracts from mouse cells. Preliminary characteriza- 
tion showed that this kinase activity phosphorylates 
serine residues in the CTD. Both ATP and GTP can 
serve as phosphate donors. With this peptide sub- 
strate assay, a protein kinase that phosphorylates 
the CTD was purified from mouse ascites tumor 
cells. 
The purified enzyme contains subunits of 58 and 
34 kDa. The 34 kDa subunit was isolated and di- 
gested with trypsin, and several peptides were puri- 
fied and sequenced. Comparison of p34 peptide se- 
quences to protein sequence data banks revealed 
that p34 is the mouse homologue of the yeast cell- 
cycle control protein cdc2. The cdc2 protein kinase 
and its budding yeast homologue CDC28 have 
been implicated in the transitions between Gl and 
S phase and between G2 and M phase of the cell 
cycle. The discovery that cdc2 is part of a complex 
that can phosphorylate RPII suggests that phos- 
phorylation of the CTD may play some role in cell- 
cycle control and that transcription may be regu- 
lated in a cell-cycle-dependent fashion. Another 
implication of the involvement of cdc2 in transcrip- 
tional regulation is that CTD kinase may be part 
of the signal transduction pathway that links cel- 
lular responses at the cell surface to changes in 
gene expression. The cdc2 protein kinase is itself 
a phosphoprotein and may be phosphorylated 
by proto-oncogene products like c-src and c-mos. 
Therefore changes in the signal transduction 
pathway brought about by oncogenic transforma- 
tion may exert their effects on gene expression 
through CTD kinase. Current studies are aimed at 
characterizing the CTD kinase subunits and devel- 
oping in vivo and in vitro systems for studying the 
role of CTD phosphorylation in the transcription 
process. 
One approach has been to purify the CTD and 
use the detached domain in biochemical experi- 
ments designed to look for interactions between 
this domain and components of the transcriptional 
apparatus. The CTD has been overexpressed in 
both prokaryotic and eukaryotic expression vector 
systems, and the resulting proteins have been puri- 
fied. The CTD expressed in eukaryotic cells is 
highly phosphorylated. The structures of both the 
phosphorylated and unphosphorylated CTDs and 
their interactions with other transcription compo- 
nents are being analyzed. 
A genetic approach developed in Dr. Corden's 
laboratory enables the reintroduction by trans- 
fection of RPII genes with alterations in the CTD 
coding sequence. This approach has been made 
possible by cloning and characterizing a-amanitin- 
resistance alleles of the gene of the largest RPII sub- 
unit. Such amanitin-resistance genes can be trans- 
fected into tissue culture cells, where they confer 
resistance to amanitin. Secondary mutations in the 
CTD of the amanitin-resistance gene can be co- 
selected, and its effect on the ability to confer 
amanitin resistance can be tested. This approach 
has shown that large deletions in the CTD are le- 
thal, implying that the CTD plays an essential role 
in the transcription process. Smaller deletions and 
point mutations have recently been constructed 
and reintroduced into cells. The effects of these 
mutations on the ability of RPII to transcribe prop- 
erly are being analyzed. 
In collaboration with the laboratory of Dr. Paul A. 
Overbeek (HHMI, Baylor College of Medicine), 
transgenic mice harboring a-amanitin-resistant RPII 
were recently developed to analyze mutations in 
RPII. These mice are resistant to high concentra- 
tions of amanitin; under these conditions transcrip- 
Continued 
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