Structural Studies of Protein-Nucleic Acid 
Interactions 
Thomas A. Steitz, Ph.D. — Investigator 
Dr. Steitz is also Professor of Molecular Biophysics and Biochemistry and of 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 fane 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 f. Abelson at the California Institute of Technology. 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 
other 62 possible anticodons. The structural 
bases of Gln-tRNA synthetase recognition are 
currently being pursued by determining the 
structures of mutant tRNAs complexed with the 
enzyme. 
The protein domain that contains the active 
site has a structure similar to that of the homolo- 
gous domains of the tyrosyl- and methionyl-tRNA 
synthetases and by amino acid sequence similar- 
ity is homologous to another 10 of the 20 synthe- 
tases. Synthetases for 10 amino acids belong to a 
second unrelated class of synthetases. This work 
is supported by a grant from the National Insti- 
tutes of Health. 
Regulation of Gene Expression 
In Escherichia colt 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 both a 30-bp DNA fragment 
and cAMP. The earlier CAP • cAMP structure had 
shown each subunit of this dimer to consist 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, in the first com- 
plex the DNA is bent, with an overall bend of 
about 90° . In both complexes most of the bend is 
achieved by two large kinks of about 43° each. 
The relationships between this CAP-induced DNA 
bend and transcription activation are presently 
being pursued by attempts to crystallize CAP with 
polymerase and DNA. 
393 
