RNA CATALYSIS AND THE STRUCTURE OF CHROMOSOME ENDS 
Thomas R. Cech, Ph.D., Investigator 
The goal of Dr. Cech's laboratory is to contribute 
to the understanding of gene expression and chro- 
mosome structure in eukaryotes. In the area of 
gene expression the focus is on RNA splicing, par- 
ticularly self-splicing, in which the folded structure 
of the RNA catalyzes the rearrangement of phos- 
phodiester linkages. The ribosomal RNA intron 
from the unicellular eukaryote Tetrahymena ther- 
mophila provided the first example of RNA self- 
splicing and continues to be the major experimen- 
tal system in Dr. Cech's laboratory The objective is 
to understand both the mechanism of RNA catalysis 
and the structure of the active site of the molecule. 
In the area of chromosome structure, DNA-pro- 
tein interactions at telomeres, the natural ends of 
linear chromosomes, are being studied. Oxytricha 
nova, another protozoan, is particularly rich in 
telomeres, with ~1 million- fold more chromo- 
somes per nucleus than a human cell. This has fa- 
cilitated the purification of the telomere-binding 
protein. The aim is to understand first how the pro- 
tein recognizes the repeated T^G^ sequence at the 
chromosome ends and then how the protein inter- 
acts with the DNA replication machinery Human 
telomeres are similar at the DNA level, consisting of 
a repeated T^AG^ sequence; thus the findings in 
Oxytricha may be generally applicable. 
I. RNA Catalysis. 
A. Reverse self-splicing of the Tetrahymena intron. 
Incubation of ligated exon RNA with the linear in- 
tron produced a molecule in which the splice-site 
sequences of the precursor RNA were re-formed. 
Integration of the intron into ligated exon sub- 
strates that have the ability to form stem-loop struc- 
tures was reduced at least one order of magnitude 
over short, unstructured substrates. This led to the 
proposal that the formation of such structures 
helps drive splicing, an intrinsically reversible reac- 
tion, in the forward direction. Integration of the 
Tetrahymena intron into a P-globin transcript also 
occurred in vitro; this result has implications for 
transposition of group I introns. 
B. Stereochemistry of RNA cleavage by the Tetrahy- 
mena ribozyme. Shortened versions of the Tetrahy- 
mena intron act as RNA enzymes, or ribozymes. 
One of the ribozyme activities is that of a sequence- 
specific endoribonuclease. This system facilitates 
study of the chemistry of the reaction. A single 
phosphorothioate was introduced at the cleavage 
site in the substrate RNA. Product analysis revealed 
that the reaction proceeds with inversion of config- 
uration at phosphorus, consistent with an in-line, 
Sj^2 (P) mechanism. Thus the ribozyme reaction is 
in the same mechanistic category as the individual 
displacement reactions catalyzed by protein nucle- 
otidyltransferases and nucleases. 
C. Fidelity of RNA cleavage by the Tetrahymena 
ribozyme. Specificity of cleavage is determined by 
base-pairing between the active site of the ribozyme 
and its RNA substrate. Surprisingly, single-base 
changes in the substrate RNA that give a mis- 
matched substrate-ribozyme complex enhance the 
rate of cleavage. The mechanistic explanation is 
that mismatches accelerate a rate-limiting product- 
release step. Addition of a destabilizing agent (urea 
or formamide) reverses the substrate specificity, al- 
lowing the ribozyme to discriminate against mis- 
matched substrates. 
D. Defining the inside and outside of the 
ribozyme. Fe(II)-EDTA, a solvent -based reagent that 
cleaves both double- and single-stranded RNA, was 
used to investigate the structure of the Tetrahy- 
mena ribozyme. Most of the catalytic core is pro- 
tected from cleavage. The data provide experimen- 
tal evidence that an RNA enzyme, like a protein 
enzyme, has an interior and an exterior. The tech- 
nique is expected to be informative for probing the 
tertiary structure of RNA molecules. 
II. DNA-Protein Interactions at Telomeres. 
A. Properties of the protein. Glycerol gradient sedi- 
mentation and protein-protein crosslinking indi- 
cated that the 43 and 5 5 kDa polypeptides are sub- 
units of a heterodimer. Both subunits are very basic 
(pi > 8.5). 
B. Assembly and self-association of telomeric com- 
plexes. The protein was found to bind to the 3' end 
of single-stranded oligonucleotides that have the 
telomeric sequence (T^G^)^, where n >1, reconsti- 
tuting the methylation protection seen with 
macronuclear DNA. Three oligonucleotide-protein 
complexes were resolved by nondenaturing gel 
electrophoresis; all were specific for the telomeric 
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