Structural Studies of Protein-Nucleic Acid 
Interaction 
Thomas A. Steitz, Ph.D. — Investigator 
Dr. Steitz is also Professor of Molecular Biophysics and Biochemistry and Chemistry at Yale University. He 
received a B.A. degree in chemistry from Lawrence College in Appleton, Wisconsin, and a Ph.D. degree in 
molecular biology and biochemistry from Harvard University, with W. N. Lipscomb. After a postdoctoral 
year at Harvard, he moved to the MRC Laboratory of Molecular Biology in Cambridge, England, as a Jane 
Coffin Childs fellow, with D. M. Blow. He next joined the Yale faculty, where he has remained, except for 
sabbatical work with K. Weber in Gottingen, West Germany, with A. Klug at Cambridge, and with J. 
Abelson at Caltech. He has received the Pfizer Prize of the American Chemical Society and is a member of 
the National Academy of Sciences. 
OUR general long-term goal is to determine 
the detailed molecular mechanisms by 
which those proteins and nucleic acids that are 
involved in the central dogma of molecular biol- 
ogy (DNA replication, transcription, and transla- 
tion) achieve their biological functions. Virtually 
all aspects of the maintenance and expression of 
information stored in the genome involve inter- 
actions between proteins and nucleic acids. We 
are seeking to provide a structural and chemical 
basis for these fundamental processes. 
Synthetase-tRNA Complex 
Enzymes called aminoacyl-tRNA synthetases 
translate the genetic code by attaching the 
correct amino acid to a tRNA containing the ap- 
propriate anticodon. Of significant current inter- 
est is how these synthetases can accurately distin- 
guish among the 60 or so similar tRNA molecules. 
Furthermore, how does RNA recognition by a 
protein differ from DNA recognition? Finally, can 
the structure of the present-day synthetase-tRNA 
complex provide any insights into the evolution 
of this central process and the evolution of the 
genetic code itself? 
We have determined the crystal structure of 
glutaminyl-tRNA synthetase (GlnRS), a 64,000- 
molecular- weight monomeric protein, com- 
plexed with tRNA^'" and ATP. The GlnRS consists 
of four domains arranged to give an elongated 
molecule that interacts with the inside of the L- 
shaped tRNA from its anticodon to its acceptor 
end. GlnRS specifically recognizes the correct 
tRNA by interactions with the three bases of the 
anticodon and with base pairs of the amino acid 
acceptor stem of the tRNA. The three bases of the 
anticodon are unstacked and splayed out; each 
base binds into a separate recognition pocket on 
the enzyme. The extensive hydrogen-bonding in- 
teractions between the protein and the anticodon 
bases make the enzyme specific for the two gluta- 
mine anticodons (UUG and CUG) but none of the 
62 other possible anticodons. 
The protein domain that contains the active 
site has a structure similar to that of the analogous 
domains of the tyrosyl- and methionyl-tRNA syn- 
thetases and similar to another 7 of the 20 synthe- 
tases. Synthetases for 10 amino acids belong to a 
second unrelated class of synthetases. 
Regulation of Gene Expression 
In Escherichia coli a reduction in glucose con- 
centration results in a rise in the levels of a sec- 
ond-messenger molecule, cAMP, and subse- 
quently to an increase in the proteins that 
metabolize other sugars. This is achieved because 
cAMP binds to the catabolite gene activator pro- 
tein (CAP), which in turn binds to specific se- 
quences at transcription start sites, activating the 
transcription of the catabolite genes. We wish to 
know how the binding of cAMP promotes the se- 
quence-specific DNA binding of CAP and how 
this binding then activates the transcribing en- 
zyme, RNA polymerase. 
We have now determined the structure of CAP 
cocrystallized with a 30-bp DNA fragment and 
cAMP. The earlier CAP • cAMP structure had 
shown that each subunit of this dimer consists of 
two domains, the larger of which binds cAMP. 
The two small domains are seen to bind DNA with 
the helix-turn-helix interacting in the major 
groove as anticipated. Strikingly, this complex 
shows a severely bent DNA, with an overall bend 
of about 90°. Most of the bend is achieved by two 
large kinks of about 40° each. The relationships 
between this CAP-induced DNA bend and tran- 
scription activation are presently being pursued. 
Replication of DNA 
E. coli DNA polymerase I functions primarily in 
the repair of DNA but is homologous to polymer- 
ases involved in replication. We have determined 
the structure of the Klenow fragment, a portion 
of Pol I that retains the polymerase and a 3 - to 
5'-editing exonuclease activity. We have shown 
that another structural domain, with a cleft large 
enough to bind duplex DNA, contains the active 
site for the polymerase reaction, whereas a 
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