Structure and Function ofRNA Polymerase 11 
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 transfer of genetic infor- 
mation from DNA to cell components is the 
synthesis of an RNA copy of the gene. This pro- 
cess is termed transcription, and the RNA synthe- 
sized is called a messenger RNA (mRNA) . The pro- 
cess can be thought of as making a copy of part of 
the architectural plan for the construction of a 
building. The synthesis of each protein compo- 
nent of the cell is directed by a distinct mRNA 
molecule, and these mRNA molecules are all syn- 
thesized by an enzyme called RNA polymerase II. 
This enzyme recognizes each gene and synthe- 
sizes the appropriate amount of mRNA at the re- 
quired time. 
RNA polymerase II contains more than 1 0 dif- 
ferent proteins whose precise functions are 
largely unknown. Our goal is to understand the 
structure and function of the subunits of RNA 
polymerase II. Six years ago my laboratory began 
to analyze the largest subunit of the mouse RNA 
polymerase II complex. 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 enzymatic synthesis of RNA. We 
have cloned and sequenced the mouse gene en- 
coding 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 different domains of the largest sub- 
unit. The aim of these experiments is to under- 
stand how RNA polymerase II orchestrates the or- 
dered expression 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 coli) 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 
5 2 -fold repeat of a seven-amino acid sequence. 
This unusual sequence, while absent in bacteria, 
is found in the large subunits of virtually every 
RNA polymerase II, including those of animals, 
plants, insects, and protists. We are currently fo- 
cusing 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 rein- 
troduction 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. 
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- 
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