Table Of ContentCONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors' contributions begin.
Lucy G. Andrews (237), Department of Microbiology, Duke University Medical
Center, Durham, North Carolina 27710
Patricia Ansel-McKinney (285), Department of Microbiology and Molecular
Genetics, Harvard Medical School, Boston, Massachusetts 02139; Harvard-
M.I.T. Division of Health Sciences and Technology, Massachusetts Institute
of Technology, Cambridge, Massachusetts 02139
Daron C. Barnard (55), Department of Molecular Biology, Vanderbilt University,
Nashville, Tennessee 37235
Joel G. Belasco (263), Skirball Institute of Biomolecular Medicine, Department
of Microbiology, New York University Medical Center, New York, New
York 10016
Graham J. Belsham (323), BBSRC Institute for Animal Health, Pirbright, Wok-
ing, Surrey, GU24 ONF United Kingdom
Paul R. Bohjanen (1), Department of Molecular Cancer Biology, Division of
Infectious Diseases, Department of Medicine, Duke University Medical Cen-
ter, Durham, North Carolina 27710
Michael W. Briggs (111), Department of Microbiology and Immunology, Uni-
versity of Rochester School of Medicine and Dentistry, Rochester, New
York 14642
Cheryl Y. Brown (305), Department of Biochemistry, University of Washington,
Seattle, Washington 98195
J. Scott Buffer (111), Department of Microbiology and Immunology, University
of Rochester School of Medicine and Dentistry, Rochester, New York
14642
Zbigniew Dominski (163), Program in Molecular Biology and Biotechnology,
University of North Carolina, Chapel Hill, North Carolina 27599
Billy T. Dye (55), Department of Molecular Biology, Vanderbilt University,
Nashville, Tennessee 37235
Mariano A. Garcia-Blanco ,1( 25), Department of Molecular Cancer Biology,
Division of Infectious Diseases, Departments of Medicine and Microbiology,
Duke University Medical Center, Durham, North Carolina 27710
iiix
xiv srotubirtnoC
Lee Gehrke (285), Department of Microbiology and Molecular Genetics, Harvard
Medical School, Boston, Massachusetts 02115; Harvard-M.I.T. Division of
Health Sciences and Technology, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
Paul D. Gershon (127), Department of Biochemistry and Biophysics/Institute of
Biosciences and Technology, Texas A&M University, Houston, Texas 77030
Gregory M. Gilmartin (79), Department of Microbiology and Molecular Genet-
sci and the Markey Center for Molecular Genetics, University of Vermont,
Burlington, Vermont 05405
Michael R. Green (31), Howard Hughes Medical Institute and Program in
Molecular Medicine, University of Massachusetts Medical Center, Worces-
ter, Massachusetts 01605
Aidan N. Hennigan (149), Department of Molecular Genetics and Microbiology,
University of Massachusetts Medical School, Worcester, Massachusetts 01655
Martin Holcik* (195), Howard Hughes Medical Institute, Departments of Genet-
sci and Medicine, University of Pennsylvania School of Medicine, Philadel-
phia, Pennsylvania 19104
Ognian C. lkonomoval " (371), Neurobiology Group, Worcester Foundation for
Biomedical Research, Shrewsbury, Massachusetts 01545
Michele H. Jacob:~ (371), Neurobiology Group, Worcester Foundation for Bio-
medical Research, Shrewsbury, Massachusetts 01545
Allan Jacobson (149), Department of Molecular Genetics and Microbiology,
University of Massachusetts Medical School, Worcester, Massachusetts 01655
Marty R. Jacobson (341), Cell Biology Group, Worcester Foundation for Bio-
medical Research, University of Massachusetts Medical School, Shrewsbury,
Massachusetts 01545
Chaitanya Jain (263), Skirball Institute of Biomolecular Medicine, Department
of Microbiology, New York University Medical Center, New York, New
York 10016
Sharon F. Jamison~ (25), Department of Molecular Cancer Biology, Duke
University Medical Center, Durham, North Carolina 27710
Jack D. Keene (237), Department of Microbiology, Duke University Medical
Center, Durham, North Carolina 27710
* Present :sserdda Molecular Genetics Laboratory, University of Ottawa, Children's Hospital of
Eastern Ontario, Ottawa, Ontario KIH 1L8 Canada.
t Present :sserdda Department of Psychiatry, Wayne State University, Detroit, Michigan 48201.
Present address: Department of Neuroscience, Tufts University School of Medicine, Boston,
Massachusetts 02111.
Present address: INTRONN LLC, Durham, North Carolina 27702.
srotubirtnoC xv
lte A. Laird-Offringa (211), Departments of Surgery and of Biochemistry and
Molecular Biology, University of Southern California School of Medicine,
soL Angeles, California 90033
Stephen A. Liebhaber (195), Howard Hughes Medical Institute, Departments
of Genetics and Medicine, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
Yi Liu (1), Departments of Molecular Cancer Biology and Microbiology, Duke
University Medical Center, Durham, North Carolina 27710
James G. McAfee (55), Department of Molecular Biology, Vanderbilt University,
Nashville, Tennessee 37235
Armen S. Manoukian (361), Division of Cell and Molecular Biology, Ontario
Cancer Institute, Department of Medical Biophysics, University of Toronto,
Toronto, Ontario M5G 2M9, Canada
Concepci6n Martinez (31), Gene Expression Programme, European Molecular
Biology Laboratory, D-69117 Heidelberg, Germany
William F. Marzluff (163), Program in Molecular Biology and Biotechnology,
University of North Carolina, Chapel Hill, North Carolina 27599
Cameron R. Miller (25), Department of Molecular Cancer Biology, Duke Uni-
versity Medical Center, Durham, North Carolina 27710
David R. Morris (305), Department of Biochemistry, University of Washington,
Seattle, Washington 98195
James G. Patton (55), Department of Molecular Biology, Vanderbilt University,
Nashville, Tennessee 37235
Thoru Pederson (341), Cell Biology Group, Worcester Foundation for Biomedi-
lac Research, University of Massachusetts Medical School, Shrewsbury, Mas-
sachusetts 01545
Aaron Proweller (111), Department of Microbiology and Immunology, Univer-
sity of Rochester School of Medicine and Dentistry, Rochester, New
York 14642
Hangjun Ruan (305), Department of Biochemistry, University of Washington,
Seattle, Washington 98195
Carlos Sufi~ (1), Department of Molecular Cancer Biology, Duke University
Medical Center, Durham, North Carolina 27710
Juan Valcfircel (31), Gene Expression Programme, European Molecular Biology
Laboratory, D-69117 Heidelberg, Germany
Zeng-Feng Wang (163), Program in Molecular Biology and Biotechnology,
University of North Carolina, Chapel Hill, North Carolina 27599
Michael L. Whitfield (163), Program in Molecular Biology and Biotechnology,
University of North Carolina, Chapel Hill, North Carolina 27599
Jeffrey Wilusz (99), Department of Microbiology and Molecular Genetics,
UMDNJ-New Jersey Medical School, Newark, New Jersey 07103
PREFACE
In the past decade or so, there has been a virtual explosion in the field of
RNA science. While most investigations had been limited to studying either bulk
populations ofmRNAs or, in a few cases, specific mRNAs that are highly enriched
in certain cells, such sa globin mRNA in reticulocytes, almost any laboratory can
now examine even very rare messages in any type of cell. Huge quantities of
specific mRNAs are synthesized in vitro with bacteriophage RNA polymerases,
minute quantities of specific mRNAs in single cells are measured by reverse
transcription and the polymerase chain reaction (RT-PCR), specific, localized
mRNAs are detected by in situ hybridization, and specific mRNA expression si
disrupted by ribozyme or antisense oligonucleotide-directed cleavage or by anti-
sense RNA-mediated translational silencing. And this si only the beginning! A
wonderful new array of techniques useful for the identification and cloning of
cDNAs for specific RNA binding proteins, which, of course, control mRNA
processing, transport, translation, and degradation, si available.
This volume deals with new methods used in RNA research. Although a
grouping of the chapters would be somewhat arbitrary, there si a common theme
sa to what each method can accomplish. For example, six chapters deal with the
isolation and characterization of specific RNA binding proteins, seven chapters
examine RNA metabolism and associated regulatory proteins, four chapters are
aimed at mRNA detection and localization, and two chapters take a genetic
approach to the study of RNA function.
This compilation of methods si meant to go well beyond that presented in
the primary literature. These chapters contain the procedural details that may not
be fully elaborated in .journal articles because of space limitations, details that can
often make the difference between success and failure. The volume will be useful
not only to newcomers to the RNA field but also to established investigators
whose research has led them to explore different facets of RNA biology.
Joel D. Richter
iivx
The Tat-TAR RNP, a Master Switch That
Regulates HIV-1 Gene Expression
Carlos Sufi6 Paul R. Bohjanen
Department of Molecular Cancer Biology Department of Molecular Cancer Biology
Duke University MedicCaeln ter Division of Infectious sesaesiD
Durham, North Carolina 27710 Department of Medicine
Duke University Medical Center
Durham, North Carolina 27710
Yi Liu Mariano A. Garcia-Blanco
Departments of Molecular Cancer Department of Molecular Cancer Biology
Biology and Microbiology Division of Infectious sesaesiD
Duke University MedicCaeln ter Departments of Medicine dna
Durham, North Carolina 27710 Microbiology
Duke University Medical Center
Durham, North Carolina 27710
I. INTRODUCTION
The study of the regulation of human immunodeficiency virus type 1 (HIV-
)1 gene expression has increased our understanding of the biology of complex
retroviruses and provided useful insights into eukaryotic transcriptional control.
The HIV-1 viral protein Tat increases HIV-1 transcription more than 100-fold
and si absolutely required for viral replication (Dayton et al., 1986; Fisher et al.,
1986). Tat si a protein of 86 amino acids that functions by binding to an RNA
element designated TAR (for trans-activation response element) (reviewed in Gait
and Karn, 1993; Cullen, 1995). TAR si a highly structured RNA element located
at the 5' end of lla HIV-1 transcripts and si composed of double-stranded stems,
a four-nucleotide bulge, and a six-nucleotide loop (Fig. :1 TAP.). Tat binds
specifically to the TAR bulge in vitro and on binding alters the structure of the
TAP. RNA (Colvin and Garcia-Blanco, 1992; Puglisi et al., 1992). Although the
structure of TAP. and the requirements for Tat-TAR binding have been thor-
oughly investigated, little si known about the mechanism by which Tat increases
HIV-1 transcription. Current models suggest a role for Tat in the regulation of
transcriptional initiation sa well sa elongation (reviewed in Cullen, 1993; Jones
and Peterlin, 1994; Karn et at., 1996).
1
mRNA Formation and Function
Copyright (cid:14)9 1997 by Academic Press. All rights of reproduction in any form reserved.
Figure 1 Model for transcriptional control by HIV-1 Tat. )A( Basal transcription of the HIV-1 promoter. In the absence of the
Tat-TAR RNP, basal transcription si carried out by core RNA polymerase (pol) II complexes that lack elongation-competent
components. These complexes are elongation incompetent and synthesize short transcripts that are released from the HIV-1 DNA
template. )B( Activation of the HIV-1 promoter by the Tat-TAR R3qP complex. Tat may trans-activate the HIV-1 promoter by
modifying and/or recruiting transcription components that transform elongation-incompetent RNA pol II complexes to elongation-
competent complexes that are now able to synthesize long transcripts. Part of this model si based on data by Sufi6 et al. (1997). All
factors involved in transcription initiation and elongation have not been drawn to simplify the model.
4 solraC 6ifuS te .la
Experimental data suggest that the primary role of TAR si to anchor Tat to
a location near the start site of transcription (Selby and Peterlin, 1990; Southgate
et al., 1990). Because mutations in the TAR loop that abolish Tat-mediated trans-
activation in vivo minimally affect Tat binding in vitro, it was postulated that host
proteins recognize the TAR loop (Feng and Holland, 1988; Roy et al., 1990).
Considerable genetic and biochemical evidence supports the existence of an essen-
tial cellular factor that binds to the TAR loop (Marciniak et al., 1990; Madore
and Cullen, 1993; Bohjanen et al., 1996). Although genetic studies suggest that
Tat binds to TAR sa a preformed complex in vivo (Madore and Cullen, 1993), it
remains unclear whether the cellular loop-binding factor si a component of this
Tat-containing complex or if it binds to TAR independently. Numerous cellular
proteins that bind to Tat or TAR have been identified and certain ones have been
characterized in some detail (reviewed in Gaynor, 1993), but the role of most of
these proteins remains unknown. Recent in vitro experiments have identified
proteins that bind to Tat and are essential for Tat-mediated trans-activation &ifuS(
and Garcia-Blanco, 1995a; Zhou and Sharp, 1995). It appears that Tat and cellular
factors form a complex on TAR RNA. This complex, referred to sa the Tat-TAR
ribonucleoprotein (RNP), constitutes the core of a master switch that likely regu-
lates the Tat-dependent increase in HIV-1 transcription by communicating with
an RNA polymerase II transcription complex (Fig. .)1
Complete elucidation of the mechanism by which the Tat-TAR RNP
activates HIV-1 gene transcription will require identification, purification, and
characterization of lla essential components in a reconstituted system. The establish-
ment of a cell-free system that faithfully recapitulates Tat-dependent activation of
HIV-1 transcription was an essential step toward this objective (Marciniak et at.,
1990). The in vitro transcription system has been used to characterize the functions
of Tat and TAR cofactors (Sheline et al., 1991; Wu et al., 1991; Sufi~ and Garcia-
Blanco, 1995a; Zhou and Sharp, 1995) and has provided important insights into
the mechanism by which Tat activates the HIV-1 promoter (Marciniak and Sharp,
1991; Laspia et al., 1993; Bohan et al., 1992; Kato et al., 1992; Graeble et al.,
1993). We will discuss the basis of this system and show how it can be used to
study the role of the Tat-TAR RNP in HIV-1 gene regulation.
II. METHODS
A. SYNTHESIS AND PURIFICATION OF RECOMBINANT HIV-1
TAT PROTEIN
Large quantities of pure Tat protein were needed to carry out the in vitro
experiments described in this chapter. Purified Tat protein has been obtained by
recombinant methods or chemical synthesis. Due to its poor solubility, complex
protocols of purification using strong denaturing agents have been developed
.1 Tat-TAP, PN.,P 5
(Green and Loewenstein, 1988; Chun et al., 1990; Waszak et al., 1992). Previously,
we described an easy procedure for the purification of large amounts of biologically
active recombinant HIV-1 Tat protein using the pGEX-2TK vector (Pharmacia,
Piscataway, NJ) &kluS( and Garcia-Blanco, 1995a). This vector allows expression
of the gene products sa fusions with the glutathione S-transferase (Gst) of Schistosoma
japonicum (Smith and Johnson, 1988). This plasmid encodes a specific thrombin
cleavage site to split the fusion and a high-aflfinity protein kinase phosphorylation
site that allows the labeling of the protein with T-32pATP. Since the Gst-Tat
fusion protein was poorly soluble, we have used a modification of the procedure
described by Grieco et al. (1992) to allow better solubilization. Briefly, 1 liter of
transformed BL-21 cells was grown to an 0D600 of 0.8, and expression of the
fusion proteins was induced by adding 0.1 mM isopropyl-/3-thiogalactopyranoside
(IPTG, Sigma, St. Louis, MO) for 3 hr. Cells were pelleted and resuspended in
10 ml of phosphate-buffered saline (PBS) containing 2 mM ethylenediaminetet-
raacetic acid (EDTA), 50/zg/ml phenylmethylsulfonyl fluoride (PMSF, Sigma),
1 /zg/ml leupeptin, and 1 /zg/ml pepstatin. The bacterial pellet was lysed on ice
by mild sonication and centrifuged at 12,000 g )xamr( for 10 rain at 4~ The
supernatant fraction was mixed with Triton X-100 to %1 (v/v) and kept on ice.
The pellet fraction was resuspended with 8 ml of a solution containing 1.5%
(v/v) sodium N-lauroylsarcosinate (Fluka, Ronkonkoma, NY), 25 mM triethano-
lamine, and 1 mM EDTA (pH 8.0), mixed and placed on ice for 30 rain. The
suspension was then centrifuged at 18,900 g )xamr( for 20 rain at 4~ Triton
X-100 and dithiothreitol (DTT) were added to the new supernatant fraction to
final concentrations of 2% and 1 mM, respectively, and the solution was mixed
with the supernatant fraction resulting from the first centrifugation. The new
solution was then rocked with 0.5 nil of glutathione-agarose beads (Sigma) for
1 hr at 4~ and the beads were washed extensively with %1 Triton X-100,
%1 Tween-20, 1 mM DTT, and 50/zg/ml PMSF in PBS. After the beads were
washed with 50 mM Tris HC1 (pH 8.0), Gst-fusion proteins were released with
10 mM glutathione (Sigma). Cleavage with thrombin (Sigma) was done with the
Gst-fusion on the beads or in solution. This reaction was carried out in cleavage
buffer (150 mMNaC1, 50 mMTris pH 8.0, 2.5 mMCaC12, 1 mMDTT) containing
10 NIH U per milliliter of thrombin for 20 rain at room temperature. The reaction
was stopped by adding 100/zg/ml PMSF. As shown in Figure 2, Gst-Tat and
Tat proteins were purified to near homogeneity. The yields of HIV-1 Tat were
up to 300/zg per liter of bacterial culture.
B. In Vitro TRANSCRIPTION SYSTEM AND HIV-1 TAT
Trans-AcTIVATION
The development of transcription-competent, cell-free systems has facilitated
the study of the complex interactions that regulate gene expression and has been
6 solraC 6ifuS te .la
Figure 2 edimalyrcaylop-SDS leg siserohportcele )EGAP-SDS( leg gniwohs the noitacifirup of recom-
binant Gst-Tat dna proteins. Tat :tfeL lane ,1 tnatanrepus the first following noitagufirtnec after gnisyl
the ;sllec lane ,2 pellet the following first centrifugation after the lysing ;sllec lane ,3 eluate from
esoraga-enoihtatulg sdaeb pure showing Gst-Tat Right: protein. lane ,1 tsG glutathione free with eluted
retfa thrombin ;noitsegid lane ,2 esoraga-enoihtatulg thrombin following eluate egavaelc pure showing
Tat protein. Gels were with stained Coomassie blue. Lane M prestained shows molecular weight
sdradnats )mahsremA( desserpxe sa ezis ni .snotladolik The snoitisop of (Gst-Tat, proteins the purified
,tsG dna Tat) era indicated yb .sworra
critical in the identification of general transcription factors. In particular, we and
others have used a previously described in vitro transcription system (Marciniak et
al., 1990) to faithfully recapitulate the Tat- and TAR-dependent transcriptional
activation of HIV-1 genes.
The in vitro transcription activity of the HIV-1 promoter is exceptionally
high in nuclear extract alone, masking the activation effect of Tat. Marciniak et
al. (1990) were the first to overcome this problem by introducing a 30-min
incubation time prior to the addition of labeled uridine triphosphate (UTP) in the
transcription reaction. Under these conditions, basal transcription was diminished
and strong activation of HIV-1 transcription was detected on addition of Tat
(Marciniak et al., 1990). Kato et al. (1992) developed a cell-free trans-activation
system with no preincubation. They found that the addition of sodium citrate at
6-8 mM in the transcription reaction decreased the basal level of the transcription,
which enabled them to observe trans-activation by Tat. We have reproduced this
effect and found that 4 mM of sodium citrate is the best concentration in our
