MACROMOLECULES OF THE CENTRAL DOGMA OF MOLECULAR BIOLOGY 
Thomas A. Steitz, Ph.D., Investigator 
Single-crystal x-ray crystallography combined 
with molecular genetics provides powerful tools for 
understanding the relationships between the struc- 
ture of a macromolecule and its biological function. 
Dr. Steitz has been using these tools to elucidate 
the structure and function of proteins and nucleic 
acids, particularly those that are involved in DNA 
replication, DNA recombination, the regulation 
of transcription, and some aspects of protein 
synthesis. The major achievements of the past year 
include the determination of the structure of 
tRNA'^'"-synthetase complex and significant prog- 
ress toward the structure determination of the Es- 
cherichia coli reck, CAP-DNA complex, and Klenow 
fragment-DNA complex. 
L Protein-DNA Interactions. 
The catabolite gene activator protein (CAP) from 
E. coli is a dimer of 22,500-molecular-weight 
subunits that activates transcription from certain E. 
coli operons in the presence of cAME This protein 
is the first of the sequence-specific DNA-binding 
proteins whose crystal structure was determined; 
each subunit was found to consist of a domain that 
binds cAMP and a smaller domain involved in bind- 
ing the DNA. 
o 
X-ray data at 3.0 A resolution from CAP cocrys- 
tallized with a 31 bp DNA fragment confirm the 
overall aspects of the CAP-DNA model built earlier 
using electrostatic calculations and other consider- 
ations. The positions of BrdU residues at each end 
of this DNA are close to those predicted from the 
model and show that the DNA is sharply bent 
around CAP, with a total bend angle of —140°. 
Complete determination of this crystal structure is 
in progress. 
Resolvase is a site-specific recombination protein 
from the transposable element gamma-delta. A few 
years ago Dr. Steitz and Dr. Nigel Grindley demon- 
strated that this protein contains a 43-amino acid 
carboxyl-terminal domain that binds to DNA in a se- 
quence-specific fashion and a 140-amino acid 
amino-terminal domain that forms the oligomeric 
interactions and catalyzes the recombination reac- 
tion. Large, single crystals were grown of both the 
amino-terminal catalytic domain and the intact pro- 
tein that diffract to —2.4 and 3.5 A resolution, re- 
spectively. 
Two mutant proteins that each contain a cysteine 
residue introduced to provide heavy-atom-binding 
sites were used to solve the structure of the 
catalytic domain. This immediately provided an un- 
derstanding of mutations that abolish recombina- 
tion without affecting DNA binding. Cocrystals of 
the intact protein with a 30 bp DNA fragment have 
been obtained, and attempts to cocrystallize with 
the 120 bp res site are in progress. 
E. coli reck protein plays a major and essential 
role in general recombination. Although crystals of 
reck were grown 10 years ago, a high-resolution 
electron density map showing features of the pro- 
tein was only achieved in the past six months. One 
additional heavy-atom derivative will be required to 
solve this structure completely. 
The large proteolytic fragment (Klenow frag- 
ment) of DNA polymerase I of E. coli was crystal- 
lized and its structure determined in Dr. Steitz's 
laboratory several years ago. This 68,000-molecular- 
weight protein catalyzes both the DNA polymerase 
and a 3 -5' exonuclease reaction. Dr. Steitz and his 
colleagues have proven that the polymerase active 
site resides on the larger carboxyl-terminal domain 
and the exonuclease active site resides on the 
smaller amino-terminal domain. 
Mutant Klenow fragment proteins have been 
made in collaboration with Dr. Grindley and Dr 
Catherine M. Joyce. A change of Asp-424 to Ala or 
of both Glu-355 and Asp-357 to Ala produces 
Klenow fragment proteins that retain full polymer- 
ase activity but exhibit no exonuclease activity. 
High-resolution crystal structure analyses of these 
proteins show that the mutations produce no de- 
tectable change in the protein structure but alter 
the binding of essential metal ions at the exo- 
nuclease active site. Structural and functional stud- 
ies of other site-directed mutant proteins altered in 
both the polymerase and exonuclease active sites 
are being pursued. 
Two cocrystal forms of Klenow fragment com- 
plexed with DNA have been grown— one with 
DNA bound at the exonuclease active site and 
one with DNA at the polymerase active site. The 
structure of the editing complex shows that the 
enzyme has denatured the duplex DNA and bound 
four single-stranded nucleotides to the 3 '-5' exo- 
nuclease active site. An electron density map of 
the DNA complex at the polymerase active 
site shows a repositioning of protein around the 
DNA-binding cleft, including a portion that is dis- 
Continued 
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