Structure and Function of RNA Polymerase II 
Jeffry L. Corden, Ph.D. — Associate Investigator 
Dr. Corden is also Associate Professor of Molecular Biology and Genetics at the Johns Hopkins University 
School of Medicine. He received his B.S. and Ph.D. degrees in biochemistry and biophysics from Oregon 
State University. His postdoctoral work was done with Pierre Chambon in the Laboratoire de Genetique 
Moleculaire des Eucaryotes at the Faculte de Medecine, Strasbourg, France. 
THE first step in the expression of genetic in- 
formation is carried out by RNA polymer- 
ase II. How this enzyme determines which genes 
are to be expressed in which cells and under 
which physiological conditions remains largely 
unknown. 
RNA polymerase II contains more than 10 dif- 
ferent proteins whose precise functions are only 
now beginning to be addressed. The goal of my 
laboratory is to understand the structure and 
function of the subunits of RNA polymerase II. 
Several years ago we began to analyze the largest 
subunit of the mouse RNA polymerase II com- 
plex. This subunit comprises one-half of the mass 
of the enzyme and, through the work of many 
laboratories, is known to be involved in the enzy- 
matic synthesis of RNA. We have cloned and se- 
quenced the mouse gene encoding this largest 
subunit and have also isolated and characterized 
several mutations in this gene. These mutant 
genes are being used to study the function of dif- 
ferent domains of the largest subunit. The aim of 
these experiments is to understand how RNA 
polymerase II orchestrates the ordered expres- 
sion of 100,000 genes during the vertebrate life 
cycle. 
The gene encoding the largest subunit of RNA 
polymerase II comprises 28 segments (exons) 
that cover 30,000 bases of mouse chromosomal 
DNA near the center of chromosome 1 1 . The 
amino acid sequence deduced from the DNA se- 
quence has revealed two interesting properties of 
the subunit. The major portion of the protein is 
similar in sequence to a bacterial RNA polymer- 
ase subunit (from Escherichia coif) that carries 
out an equivalent function. This evolutionary 
conservation is much stronger than had been ex- 
pected and has allowed us to predict that the 
mouse subunit is involved in the transcription 
elongation process. 
Although the major part of the largest subunit is 
related to the bacterial enzyme, our DNA se- 
quence analysis has also revealed a domain that is 
unique to RNA polymerase II. This domain is lo- 
cated at one end of the molecule and constitutes a 
52-fold repeat of a seven-amino acid sequence. 
This unusual sequence, although absent in bacte- 
ria, is found in other large subunits of virtually 
every RNA polymerase II, including those of ani- 
mals, plants, insects, and protists. We are 
currently focusing our efforts on understanding 
the role of this domain in the process of 
transcription. 
Our genetic approach to the function of this 
carboxyl-terminal domain (CTD) has grown from 
analysis of mutations in the largest subunit gene. 
We first isolated mutant mouse tissue-culture cell 
lines that are resistant to the mushroom toxin a- 
amanitin. The largest subunit genes from several 
of these cell lines have been cloned and, by re- 
introduction into amanitin-sensitive cells, have 
been shown to confer resistance to a-amanitin. 
We have used this gene transfer technique to 
map the mutations responsible for amanitin 
resistance. 
The availability of a well-defined, selectable ge- 
netic marker in the largest subunit gene has 
proved useful in the analysis of the role of the 
CTD. Deletion, insertion, and substitution muta- 
tions have been created in the CTD of an amani- 
tin-resistance gene. The effect of these mutations 
has been tested by introducing the altered resis- 
tance genes into cells and scoring for amanitin 
resistance. Removing the entire CTD eliminates 
the ability to transfer amanitin resistance, demon- 
strating that the CTD plays an essential role in 
transcription. We have also shown that up to 20 
of the 52 repeats are dispensable for growth in 
tissue culture, indicating either that the CTD is 
functionally redundant or that dispensable re- 
peats are only necessary in some cell types. Para- 
doxically, we have recently shown that the CTD 
is highly conserved among all mammals. We 
are now designing experiments to assess the role 
of dispensable heptapeptide repeats during 
development. 
We have also been examining postsynthetic 
modifications of the CTD. This domain is rich 
in amino acids (such as serine, threonine, and 
tyrosine) that can be modified by addition of 
phosphate groups. RNA polymerase II is a phos- 
phorylated enzyme, but no function for phos- 
phorylation is known. We have used synthetic 
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