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PROGRESS IN
Nucleic A c i d Research
a n d M o l e c u l a r Biology

edited by

KlVlE MOLDAVE
Department of Molecular Biology and Biochemistry
University of Calijimia, Irvine
lrvine, Calijiiiu

Volume 58

ACADEMIC PRESS
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Contents

SOME~ T I C L E SPLANNED VOLUMES...............

FOR FUTURE ix

The Hairpin Ri bozyme: Discovery. Two-Dimensional

Model. and Development for Gene Therapy . . . . . . . . . 1
Arnold Hampel
I. Discovery ................................................... 5
I1. Biochemical Properties ........................................ 7
I11. The Hairpin Ribozyme Model .................................. 10
IV. Development for Gene Therapy ................................ 15
V. Delivery of the Hairpin Ribozyme for Gene Therapy ............... 18
VI. Inhibition of HIV-1 Expression in Viuo ........................... 20
VII. Additional Hairpin Ribozymes-GUA Specific ..................... 33
.
VIII Conclusions and Perspectives ................................... 36
References ................................................... 38

Serum- and Polypeptide Growth Factor-Inducible

Gene Expression in Mouse Fibroblasts . . . . . . . . . . . . . 41
Jeffi-ey A . Winkles
I. Mitogenic Stimulation of Quiescent Fibroblasts:
The Genomic Response ....................................... 43
I1. Identification of Serum- and Polypeptide Growth Factor-Inducible
Genes: Strategies and Results ................................... 48
I11. Serum- and Polypeptide Growth Factor-InducibleGene Products
and the Control of Cellular Proliferation ......................... 60
IY Conclusions ................................................. 69
References ................................................... 70

Regulation of Translational Initiation during

Cellular Responses to Stress . . . . . . . . . . . . . . . . . . . . 79
Charles 0. Brostrom and Margaret A Brostrom .
I. Stress Responses and Stress Proteins of Eukaryotic Cells ............ 82
I1. Regulation of TranslationalInitiation ............................ 90

V
vi CONTENTS

111. Translational Accommodation to ER or Cytoplasmic Stress ......... 110

IV. Perspectives and Speculation ................................... 116
References ................................................... 120

Lactose Repressor Protein: Functional Properties

and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Kathleen Shive Matthews and Jeffry C. Nichols
I. Lactose Repressor Protein ..................................... 130
I1. DNABinding ................................................ 134
111. Inducer Binding .............................................. 139
N . Structure and Function ........................................ 142
V. NMR and X-ray Crystallographic Structures ...................... 149
VI. Applications of Lac1 Control ................................... 155
VII. Conclusion and Prospects for the Future ......................... 156
References ................................................... 157

Copper-Regulatory Domain Involved

in Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Dennis R . Winge
I. Copper Ion Sensing in Prokaryotes .............................. 168
I1. Copper Sensing in Eukaryotes .................................. 169
I11. Copper Metalloregulation in Yeast ............................... 170
IV. Metal Clusters in Regulation ................................... 188
V. Summary and Perspective ...................................... 190
References ................................................... 191

Molecular Biology of Trehalose and the Trehalases

in the Yeast Saccharomyces cerevisiae . . . . . . . . . . . . . 197
Solomon Nwaka and Helmut Holzer
I. Metabolism of Trehalose in Yeast ................................ 199
I1. Biological Functions of Trehalose in Yeast ........................ 202
111. Characterization and Location of the Yeast Trehalases .............. 207
IV. Molecular Analysis of the Yeast Trehalases ........................ 211
V. Biological Functions of the Trehalase Genes ...................... 226
VI. Trehalases and Heat Shock Proteins ............................. 229
CONTENTS vii

VII. Outlook on the Biotechnological Importance of Trehalose

and the Trehalases ............................................ 231
References ................................................... 233

Molecular and Structural Features of the Proton-Coupled

Oligopeptide Transporter Superfamily . . . . . . . . . . . . . 239
You-Jun Fei. Vadivel Ganapathy. and Frederick H. Leibach
I. Two Different Peptide Transporter Subfamilies: A Comparison
between the Members of the ABC Peptide Transporter
Subfamily and the POT Subfamily .............................. 241
I1. Molecular Cloning Procedures Employed for Identification
of the POT Family Members ................................... 243
I11. Comparison of Amino Acid Sequences of the Members
of the POT Family ............................................ 248
IV. Topological Features of the POT Subfamily ....................... 256
V. Conclusion .................................................. 257
References ................................................... 259

Doublestrand Break-Induced Recombination

in Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Fekret Osman and Suresh Subramani
I. Models of Double-Strand Break-Induced Recombination ........... 266
I1. Double-Strand Break-Induced Mitotic Recombination .............. 277
I11. Double-Strand Break-Induced Meiotic Recombination ............. 288
IV. The Genetic Control of Double-Strand Break-Induced
Recombination ............................................... 291
V. Concluding Remarks .......................................... 295
References ................................................... 295

Impaired Folding and Subunit Assembly as Disease

Mechanism: The Example of Medium-Chain
acyl-CoA Dehydrogenase Deficiency . . . . . . . . . . . . . . 301
Peter Bross. Brage S. Andresen. and Niels Gregersen
I. Protein Folding and Its Disturbance by Missense Mutations ......... 303
I1. The Role of MCAD in Mitochondrial P-Oxidation of Fatty Acids ..... 310
I11. Studies on the Molecular Pathology of MCAD Deficiency ........... 312
viii CONTENTS

IV. Conclusions ................................................. 327

References ................................................... 332

Interaction of Retroviral Reverse Transcriptase with

Template-Primer Duplexes during Replication . . . . . . . . 339
. .
Eric J Arts and Stuart F.J Le Grice
I. Human Immunodeficiency Virus Reverse Transcriptase ............ 341
I1. tRNDLYS*3-Mediated Initiation of (-) Strand DNA Synthesis ........ 346
I11. Interaction of RT with the Template-Primer Duplex ............... 361
IV. The RNase H Domain and Hydrolysis of RNA-DNA Hybrids ....... 370
V. The Polypurine Tract and Second-StrandSynthesis ................. 380
VI. Conclusions ................................................. 386
References ................................................... 387

INDEX ..................................................... 395

Some Articles Planned for Future Volumes

Structure and Transcription Regulation of Nuclear Genes for the Mouse

Mitochondria1 Cytochrome c Oxidase
G. AVADHANIet al.
NARAYAN
Tissue Transglutaminase: Retinoid Regulation and Gene Expression
PETERJ. A. DAVIESAND SHAKIDMIAN
Genetic Approaches to Structural Analysis of Membrane Transport Systems
WOLFGANG
EPSI-EIN
Intron-encoded snRNAs
J. FOURNIER
MAURILLE AND E. STUART
MAXWELL
Molecular Analyses of Metallothionine Gene Regulation
LASHITEW GEDAMU
et al.
Mechanisms for the Selectivity of the Cell's Proteolytic Machinery
ALFXEDGOLDBERG,
MICHAELSHERMAN,
AND OLIVERCoux

Mechanisms of RNA Editing

L. HAJDUK
STEPHEN AND SUSAN
MADISON-MNUCCI
Structure/Function Relationships of Phosphoribulokinase and Ribulosebisphosphate
Carboxylase/Oxygenase
FRED
C. HARTMAN
AND HILLELK. BRANDES

The Nature of DNA Replication Origins in Higher Eukaryotic Organisms

JOELA. HUBERMAN C. BURHANS
AND WILLIAM

Synthesis of DNA Precursors in lactobacillus acidophilus R-26

IKEDA
DAVIDH. IVESAND SEIICHIRO
A Kaleidoscopic View of the Transcriptional Machinery in the Nucleolus
T. JACOB
SAMSON
Sphingomyelinases in Cytokine Signaling
MARTIN
KRONKE
Mammalian DNA Polymerase Delta: Structure and Function
Y. W. T. LEE
MARIEITA
DNA Helicases: Roles in DNA Metabolism
STEVEN W. MATSONAND DANIELW. BEAM

ix
X SOME ARTICLES PLANNED FOR FUTURE VOLUMES

Haparan Sulfate-Fibroblast Growth Factor Family

WWACE L. MCKEEHANAND M ~ w KAN
o
Molecular Biology of Snake Toxins: Is the Functional Diversity of Snake Toxins
Associated with a Mechanism of Accelerated Evolution?
ANDREMENEZet al.
lnosine Monophosphate Dehydrogenase: Role in Cell Division and Differentiation
BEVERLYS. MITCHELL
Specificity of Eukaryotic Type II Topoisomerase: Influence of Drugs, DNA Structure,
and local Sequence
MARKT. MULLERAND JEFFREY SPITZNER
localization and Movement of tRNAs on the Ribosome during Protein Synthesis
KNUDH. NIERHAUS
lmmunoanalysis of DNA Damage and Repair Using Monoclonal Antibodies
MANFREDF.RAJEWSKY
Mechanism of Transcriptional Regulation by the Retinoblastoma Tumor Suppressor
Gene Product
PAULD. ROBBINSAND JON HOROWITZ
Organization and Expression of the Chicken &lobin Genes
AND FELIXR. TARGA
KLAUS SCHERRER
Physicochemical Studies on DNA Triplexes and Quadruplexes
RICHARDH. SHAFER
Bacillus subtilis as I Know It
NOBORUSUEOKA
Transcriptional Regulation of Steroid Receptor Genes
J.TINDALLAND M. V. KUMAR
DONALD
Molecular Genetic Approaches to Understanding Drug Resistance in Protozoan
Parasites
DYANN WIRTHd al.
The Hairpin Ribozyme:
Discovery. Two-Dimensiona I
Model. and Development
for Gene Therapy’
I ARNOLDHAMPEL
Departments of Bwlagkal Sciences
and Chemistry
Northern Illinois University
DeKalb. Illinois 60115

I. Discovery .................................................... 5
I1. Biochemical Properties ........................................ 7
111. The Hairpin Ribozyme Model .................................. 10
A . Secondary Structure ........................................ 10
B. Three-DimensionalInteractions .............................. 14
IV. Development for Gene Therapy ................................. 15
A . Targeting Rules ............................................ 15
B. Selection of Target Sites ..................................... 16
C. Design and Optimization of the Ribozyme ..................... 17
D. Catalytic Improvements ..................................... 17
V. Delivery of the Hairpin Ribozyme for Gene Therapy ............... 18
A . Autocatalytic Hairpin Cassette ............................... 18
B. Promoter ................................................. 20
VI. Inhibition of HIV-1 Expression in Vim ........................... 20
A . Targets and Ribozyme Selection .............................. 20
B. The 5‘ Leader Target and Ribozyme .......................... 21
C. The PolSpecific Target and Ribozyme ........................ 28
D. The Double Ribozyme ...................................... 31
E. Human Clinical Trials ...................................... 31
F. Improvements ............................................. 31
VII. Additional Hairpin Ribozymes-GUA Specific ..................... 33
VIII. Conclusions and Perspectives ................................... 36
References ................................................... 38

Abbreviations: 3‘F. 3’fragment; 5’F. 5’fragment;AIDS. acquired immunodeficiencysyn-

drome; bp. base pair(+ ELISA. enzyme-linked immunosorbent assay; HC. hairpin autocatalyt-
ic cassette; HIV.l. human immunodeficiency virus type 1; HN.2. human immunodeficiency
virus type 2; LTR. long terminal repeat; MMLV. Moloney murine leukemia virus; MMTV.mouse
mammary tumor virus; nt, nucleotide(s);p24 gag, one of the group-specificantigen proteins of
HIV-1; pol. RNA polymerase gene of HIV-1; RRE. rev response element in HIV-1; RT-PCR. re-
verse transcription-polymerase chain reaction; Rz.ribozyme; S. substrate; sTRSV. satellite RNA
from tobacco ringspot virus; sCYMV1. satellite RNA from chicory yellow mottle virus; sArMV.
satellite RNA from arabis mosaic virus; TAR. transcriptional activation region on HIV-1 RNA;
tat,transcriptional trans-activatorprotein of HIV.1 .

Progress in Nucleic Acid Research Copyright 0 1998 by Academic Ress.

and Molecular Biology. Vol. 58 1 AU rights of reproductionin any form resewed.
0079-6603B8 $25.00
2 ARNOLD HAMPEL

This review chronicles the discovery of the hairpin ribozyme, its charaderiza-
tion, and determination of the two-dimensional structure, culminating with its use
for human gene therapy as an AIDS therapeutic. The minimal sequence constitut-
ing the hairpin ribozyme catalytic domain was identified from a small plant viral
satellite RNA. Biochemical characterization showed it to be among the most effi-
cient of all known ribozymes. Mutagenesis determined that the two-dimensional
structure had four helices, consisting of 17 Watson-Crick base pairs and one A:G
pair for a total of 18 bp. The helices were interspersed with five single-stranded
loops. Helices 1 and 2 were located between the ribozyme and substrate, allowing
the ribozyme to recognize the substrate. The substrate had a sequence preference
of BN*GUC where * is the site of cleavage and N*GUC the substrate loop between
these two helices. By using sequences of this type, it was possible to design the ri-
bozyme to base pair with the substrate and cleave heterologous RNA substrates-
leading to design of the hairpin ribozyme for gene therapy. The HW-1 sequence was
searched for suitable target sites, and ribozymes were designed, optimized, catalyt-
ically characterized, and tested in vivo against HIV-1 targets. Two ribozymes had ex-
cellent in vitro catalytic parameters and inhibited in vivo expression of viral proteins
by 3-4 logs in tissue culture cells. viral replication was inhibited as well. They have
been developed as human AIDS therapeutics, and will likely be the first ribozymes
to be tested as human drugs in clinical trials. 8 I998 Academic Pmss

RNA catalysis was co-discoveredby S. Altman, who found the M1 RNA

of RNAse P could catalytically cleave and process the 5‘ terminus of the tRNA
precursor (I),and by T. Cech, who found the tetrahymena ribosomal RNA
intron had autocatalytic activity (2).The term “ribozyme” was coined to de-
scribe catalytic RNA. Since then, four other catalytic RNAs, all self-cleaving,
have been discovered: the hepatitis delta ribozyme, the neurospora ribozyme,
the hammerhead ribozyme, and the hairpin ribozyme. The latter two are
from plant viral satellite, virusoid, and viroid RNAs (see Ref. 3 for review).
This review focuses on the hairpin ribozyme (4,5).Specifically,I describe
its discovery-followed by the many facets of development required to bring
it to the point of being tested in clinical trials as a drug for human use as an
AIDS therapeutic. (For previous reviews of this work, see 6, 7, and for a de-
tailed description of many aspects, see 8. We have used the HIV-1 system as
an initial test model for determining the utility of the hairpin ribozyme in the
down-regulationof gene expression. Based on our excellent success with that
system, we are very optimistic that the hairpin ribozyme may have more gen-
eral utility for a wide variety of applications in other systems as well.
The hairpin ribozyme was found as the catalytic center of three known
plant satelliteRNAs. These were the negative strands of the satellite RNAs from
tobacco ringspot virus (sTRSV), chicory yellow mottle v i r u s type 1(sCYMVl),
and arabis mosaic virus (sArMV) (9,IO).Initial studies identified the hairpin ri-
bozyme first in the negative strand of sTRSV. Using molecular modeling of the
negative strand of sTRSV as a first approximation, we made substrate and ri-
THE HAIRPIN RIBOZYME 3

bozymes of various lengths and sequences in order to determine the minimum

catalytic center. A 50-nt ribozyme sequence was found to be capable of cleav-
ing a 14-nt substrate sequence in a truns reaction. It proceeded without de-
pletion of the 50-nt RNA component, and therefore was catalytic. It followed
true Michaelis-Menten kinetics, allowing determination of Km, kcat, energy of
activation, Mg2+dependence, temperature dependence, and pH optima (4.
The two-dimensional structure was determined by making an extensive
collection of site-specific mutants for both the ribozyme and the substrate.
The location of individual base pairs was determined by comparison of cat-
a l ~ activity
c for these mutant sequences with that of the native sequence.
That is, if the site of a predicted base pair lost activity with a mismatch in this
position, and if the activity was restored with an alternate base pair, then a
base pair at this site has been identified. This method identified four helices
and five loops for the ribozyme-substrate complex. The overall structure was
hairpin-like, so I named it the hairpin ribozyme ( 5 , I I ) . Of the five helices, he-
lices 1and 2 occurred between the ribozyme and the substrate, and helices 3
and 4 were within the ribozyme itself. Single-strandedloops 1,2,3, and 4 were
in the ribozyme sequence, and loop 5 was in the substrate sequence (Fig. 1).
Following its discovery, its biochemical characterization, and determina-
tion of its two-dimensional structure, the hairpin ribozyme was engineered
to cleave heterologous substrate RNAs ( 5 , I I ) .This led to development of the
hairpin ribozyme system for human gene therapy and other applications for
down-regulation of gene expression. Targeting rules for cleavage of heterol-
ogous substrates were determined. The substrate had a sequence preference
of BN*GUC where the * is the site of cleavage. The nucleotide B is G, U, or
C but not A. With these targeting rules in hand, we now had the possibility
of specifically cleaving target mRNA or viral RNA molecules, resulting in in-
hibition of gene expression or viral replication.
Sequence searches were done for a number of systems, including HIV-1,
to identdy sequences containing BN*GUC for use as possible target sites (5,
8,12,13).Using HIV-1 as an example, ribozymes were made to a number of
potential targets and in vitro cleavage efficiency of the ribozymes to these
targets carried out. Optimization was done by varying the length of helix 1
to determine its optimal length for maximum catalytic efficiency. In general
the optimal length of helix 1varied between 6 and 12 bp, with 8 bp being a
useful first approximation. Helix 2 was fixed at 4 bp.
The catalybc activity of the ribozyme was improved by making specific se-
quence changes in regions of the ribozyme containing nonessential nucleo-
tides. Certain of these changes greatly improved catalytic activity for certain
targets. Those ribozymes that had the best catalytic efficiency were used for
gene therapy in tissue culture cells. Two ribozymes, targeted to the 5’ leader
region and a region of the pol gene of HIV-1, reduced expression of HIV-1
4 ARNOLD HAMPEL

FIG.1. The hairpin ribozyme model. The negative strand of sTRSV native hairpin ri-
bozyme-substrate complex consists of a 14-nt substrate and a 50-nt ribozyme complexed to form
four helices and five single-strandedloops named and located as shown in the model (4A8J.2).
The helices consist of 18 base pairs, of which 17 are canonical Watson-Crick base pairs and 1
is a noncanonical A:G base pair (2.24.Numbering from 5’ to 3’ is 1-14 for the substrate and
1-50 for the ribozyme as standardized in the original description of the hairpin ribozyme (5).
Cleavage of the substrate occurs between A5 and G6. This model contains later modifcations
from the original model. The original model was published in both Ref. 5 and Ref. 8, and is re-
produced in modified form from the publication of the original model in A. Hampel, R. Tritz, M.
Hicks, and P. Cruz, Nucleic A& Res. 18,299 (1990) by permission of Oxford University Press.

virus by 1000 to 10,000-fold.These two ribozymes,which we developed, have

been approved by the RAC (Recombinant DNA Advisory Committee) for hu-
man use and will soon be tested as potential AIDS therapeutics in humans by
Dr. Flossie Wong-Staal at the University of California-San Diego (7).
We recently found that the hairpin ribozymes representing the catalytic
cores of the negative strands of sCYMVl and sArMV were also catalyhcally
active. These ribozymes were highly active, with catalytic efficiencies only
slightly less than that of sTRSV. Furthermore these two new classes of hairpin
THE HAIRPIN RIBOZYME 5

ribozymes had *GUA target preference, in contrast to the *GUC preference

of the sTRSV hairpin ribozyme (10).We have used the sCYMV1-based engi-
neered ribozyme to cleave two target sites in HIV-1 and one target site in hu-
man papillomavirus type 16. Thus we have essentially doubled our repertoire
of hairpin ribozymes available for targeting. This is especially important for
targeting sites in HIV-1 because it has such a high mutation rate. By generat-
ing multiple ribozymes and delivering them simultaneously, the chances of r e
ducing viral expression over the long term would be expected to be enhanced.
Details of this discovery, characterization, and development of the hair-
pin ribozyme for gene therapy follow.

I. Discovery
The phenomenon of autocatalytic cleavage and ligation of small plant vi-
roid and viral-associated RNAs had been observed to occur in cis in the large
359-nt native RNA strands (14).Others expanded upon this work to iden%
the minimal catalytic center of the positive strand of sTRSV and named it the
hammerhead ribozyme (15). Similarly, my own laboratory, utilizing these
results, carried out experiments to determine the minimum catalytic sequence
of the negative strand of sTRSV, which we named the hairpin ribozyme (4,5).
Autocatalytic cleavage and ligation were first seen for dimeric transcripts
of the 359-nt long negative strand of sTRSV (16,17).The cleaved RNA had
a 5' fragment that had a 3' terminal A and a 2',3'-cyclic phosphate, while the
3' fragment had a 5' terminal G and a newly formed 5'-OH terminus (18).
Beginning with this previous knowledge that the 359-nt negative strand of
sTRSV had catalytic activity, we began a search for the minimal sequence
that could carry out catalysis.
Figure 2 shows the catalyhc center of the negative strand of sTRSV with
the original numbering system for that of the positive strand of sTRSV (4,19).
We previously knew where the site of cleavage for the substrate was (cleav-
age occured between positions 49 and 48), and from mutagenesis experi-
ments we knew the general location of the catalytic center. Operating be-
tween positions 247 and 175 for the ribozyme (9) and 52 and 43 for the
substrate (20),we began searching for the minimal sequences required for
catalysis. We initially modeled these two regions to attempt to identdy any
regions of base pairing. We then made transcripts of the RNA components
and combined them in Cram-cleavagereactions to attempt to elicit catalysis.
We tested several of our models without success.
We observed cleavage with the minimal sequence of the catalytic center
of sTRSV in a &urn reaction (8). We later named this sequence the hairpin
ribozyme (5). The minimum sequence consisted of substrate nt 53-40, which
6 ARNOLD HAMPEL

FIG.2. The catalyhc center of the negative strand of sTRSV. The entire 359-nt sequence of
the negative strand of sTRSVwas folded into a minimum energy structure, a portion of which
is shown, and the minimum sequence determined (4,8).Numbering of the sequence is as orig-
inally described for the negative sTRSV strand (19).Shown is the active site (catalytic center of
this molecule)with the ribozyme (bottom stippled area) located between nt 224 and 175 (1-50
in parentheses numbered according to Fig. l),and the substrate (top stippled area) located be-
tween nt 53 and 40 (1-14 in parentheses numbered according to Fig. 1). The arrow marks the
site of cleavagebetween A49 and G48 (A5/G6according to the substrate numbering scheme of
Fig. 1). Reproduced from A. Hampel and R. Tritz,Biochemistry 28,4929 (1989). Copyright
1989 American Chemical Society.

corresponds to sequence 1-14 in Fig. 1, and ribozyme nt 224-175, which

corresponds to ribozyme sequence 1-50 in Fig. 1.These two sequences are
shown in Fig. 2 as the “active site,” and in Fig. 1 are modeled according to
later experimental results. When these two sequences were combined in a
trum reaction, the ribozyme gave cleavage of substrate. These were the ex-
periments that determined the minimal sequence of the catalytic center of
the negative strand of sTRSV.
Following cleavage in this reaction, we sequenced the cleavage products
of the substrate and found that, indeed, cleavage had occurred in the same
position as in the large 359-nt negative strand of sTRSV,between nt A49 and
G48.Furthermore, cleavage generated a 5’ cleavage fragment with a 2’,3’-
cyclic phosphate terminus and a 3’ cleavage fragment with a 5‘-OH termi-
nus (4,8).This showed that truns cleavage of a small substrate RNA by a por-
THE HAIRPIN RIBOZYME 7

tion of the sequence of the negative strand of sTRSV occurred at the same
cleavage site and gave the same cleavage termini as that of the large 359-nt
native sequence. The reaction is summarized as
substrate RNA: UGACA*GUCCUGUUU

I catalytic RNA

UGACA>P + HoGUCCUGUUU
5’F 3’F
products
Further attempts to reduce the number of bases in the ribozyme either
from the 5’ or 3’ terminus resulted in reduced activity (4,8).Specifically,we
removed the 3’ terminal A and the 3’ terminal UA, and then determined cat-
alytic activity (kcadK,) by measuring both kcat and K,. When the terminal
A was removed, activity was reduced fivefold.Removal of the 3’ terminal UA
reduced activity 20-fold. Similarly,when the substrate was shortened to less
than 14 nt, activity was reduced. Thus this is the minimal sequence for both
the ribozyme and substrate in the native negative strand of sTRSV.

II. Biochemical Properties

Once we had two RNA sequences that, when combined, resulted in cleav-
age of one of them, the next step was to biochemically characterize them. Us-
ing an approach similar to that successfully used for the hammerhead ri-
bozyme (24, we carried out biochemical characterizations of these two
minimal sequences from the negative strand of sTRSV (4, 8).At this point,
we only had a single-event cleavage reaction and had no idea if it was cat-
alytic, that is, if the supposed ribozyme turned over. We carried out a time
course of substrate cleavage by ribozyme using a molar ratio of substrate to
ribozyme of 30:l. The substrate cleaved to near completion (980/0),with no
loss of ribozyme during the course of the reaction (Fig. 3).This showed the
ribozyme turned over and was not used up during the the catalytic reaction,
a necessary characteristic of a biological catalyst. Furthermore, the rate of
cleavage of substrate was linearly dependent on ribozyme concentration,
again a characteristic of a biological catalyst.
Since the RNA we identifed was catalytic, we designed and carried out
kinetic analyses with ribozyme concentrationlimiting. The reaction followed
Michaelis-Menten kinetics, with initial velocity being dependent on sub-
strate concentration when ribozyme concentration was limiting. The initial
kinetic constants determined were K , of 30 nit4 and kcat of 2.l/min, which
are excellent catalytic parameters for an RNA-catalyzed reaction (Fig. 4).