cific site is expected to be generally useful for de- 
termining proximity within folded RNA molecules 
or ribonucleoprotein complexes. 
A New Class of Chemical Reactions 
Catalyzed by RNA 
All the known classes of ribozymes catalyze reac- 
tions at phosphorous centers, but no reaction at a 
carbon center had been demonstrated. The active 
site of the Tetrahymena ribozyme was engineered 
to bind an oligonucleotide derived from the 3' end 
of A'^-formyl-methionyl-tRNA*'^". This ribozyme was 
found to have a modest level of aminoacyl esterase 
activity, accelerating the hydrolysis of the fMet by as 
much as 1 5-fold beyond the uncatalyzed rate. Study 
of the ribozyme sequence requirements and magne- 
sium ion requirement for the reaction indicated that 
it takes place in the same active site responsible for 
the normal endonuclease reaction. The ability of 
RNA to catalyze reactions with amino acid substrates 
expands knowledge of the catalytic versatility of 
RNA and suggests that the original aminoacyl tRNA 
synthetase could have been an RNA molecule. 
Crystallization of Ribozymes for X-ray 
Diffraction Analysis 
Although RNA structure and transition state inter- 
actions can be inferred from biochemical experi- 
ments, physical methods such as x-ray diffraction 
and nuclear magnetic resonance (NMR) provide the 
only sure paths to obtain atomic-resolution struc- 
tures. In collaboration with Dr. Craig Kundrot (Uni- 
versity of Colorado at Boulder), a systematic ap- 
proach has been developed for crystallization of a 
number of ribozymes and structural domains 
thereof. A number of these RNAs have crystallized in 
a reproducible manner. Most current crystals are too 
small to attempt diffraction, but a l60-nucleotide 
independent folding domain of the Tetrahymena 
ribozyme has yielded crystals larger than 0. 1 mm in 
each dimension. Diffraction studies are in progress. 
(This work has also been supported by the W. M. 
Keck Foundation.) 
Engineering Ribozymes for Better Activity 
and Specificity 
The RNA-substrate-binding site of the Tetrahy- 
mena ribozyme is connected to the catalytic core by 
the joining region J 1/2. Small deletions in J 1/2 were 
found to enhance dramatically the turnover number 
and sequence specificity of the ribozyme in the en- 
donuclease reaction. Ironically, the explanation for 
these improved properties was found to lie in the 
decreased affinity of the mutant ribozyme for its 
RNA substrate, rather than improvement of the 
chemical cleavage step. (This work was supported 
by grants from the National Institutes of Health to 
Dr. Cech and from the Lucille P. Markey Charitable 
Trust to Dr. Daniel Herschlag.) 
Ribozyme Tertiary Interactions 
Involving the RNA Backbone 
In the endonuclease reaction of the Tetrahymena 
ribozyme, the helix PI, which contains the RNA 
cleavage site, must be juxtaposed to the guanosine- 
binding site. In a search for a nucleotide in the cata- 
lytic core that contributes to the stability of this ar- 
rangement, a conserved adenine (A302) was 
identified. Binding and activity studies with chi- 
meric oligonucleotides and mutant ribozymes led to 
a model in which A302 accepts an H-bond from the 
2'-OH group three nucleotides from the cleavage 
site in PI. The model was further tested and sup- 
ported by methylation footprinting experiments. 
Such base-backbone tertiary interactions may be gen- 
erally important in RNA folding. (This work was 
supported by a grant from the National Institutes of 
Health to Dr. Cech and a postdoctoral fellowship 
from the Jane Coffin Childs Fund for Medical Re- 
search to Dr. Anna Marie Pyle.) 
Human RNA- and DNA-binding Proteins 
Specific for the Telomere Repeat Sequence 
In previous work of Dr. Cech's laboratory, a pro- 
teinaceous activity was identified in HeLa cell nu- 
clear extracts that binds to single-stranded repeats of 
TTAGGG, the human telomere DNA sequence. 
More-recent studies indicated that the proteins also 
bind to the corresponding RNA sequence UUAGGG, 
with higher affinity than to DNA. Purification based 
on sequence-specific binding gave a small group of 
proteins in the 37- to 4l-kDa range. Amino acid se- 
quence analysis (Dr. Clive Slaughter, HHMI, Univer- 
sity of Texas Southwestern Medical Center at Dallas) 
identified peptides that matched known hnRNP E 
protein sequences, as well as a novel peptide 
containing the RNP 1 consensus sequence found 
in many single-stranded DNA- and RNA-binding 
proteins. Immunological reactivity (in collabora- 
tion with Dr. Gideon Dreyfuss, HHMI, University of 
Pennsylvania) identified the proteins as being re- 
lated to hnRNP D and E proteins but, interestingly, 
not the D and E proteins found in hnRNP com- 
plexes. The functional relationship of these pro- 
teins to nuclear RNA and to telomeric DNA remains 
to be determined. 
Dr. Cech is also American Cancer Society Pro- 
fessor of Chemistry and Biochemistry and of Mo- 
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