Molecular Cell 23, 297–305, August 4, 2006 ª2006 Elsevier Inc.DOI 10.1016/j.molcel.2006.06.014
Controlling the Elongation Phase
of Transcription with P-TEFb
B. Matija Peterlin1,* and David H. Price2,*
1Department of Medicine, Microbiology
Rosalind Russell Medical Research Center
University of California, San Francisco
San Francisco, California 94143
University of Iowa
Iowa City, Iowa 52242
is a cyclin-dependent kinase that controls the elonga-
tion phase of transcription by RNA polymerase II
(RNAPII). This process is made possible by the rever-
sal of effects of negative elongation factors that
include NELF and DSIF. In complex organisms, elon-
gation control is critical for the regulated expression
of most genes. In those organisms, the function of
P-TEFb is influenced negatively by HEXIM proteins
and 7SK snRNA and positively by a variety of recruit-
ing factors. Phylogenetic analyses of the components
of the human elongation control machinery indicate
that the number of mechanisms utilized to regulate
P-TEFb function increased as organisms developed
more complex developmental patterns.
RNAPII Elongation Control
Some of the most important mechanisms regulating
prokaryotic and eukaryotic gene expression target the
movement of RNA polymerases. For example, in bacte-
ria, termination, antitermination, and pausing mecha-
nisms dictate mRNA levels (Henkin and Yanofsky,
tion control mechanisms were associated with expres-
sion of c-myc, the human immunodeficiency virus
(HIV), and heat shock genes. Initial results indicated
that a block to elongation rather than initiation was re-
sponsible for repressing the c-myc gene (Bentley and
Groudine, 1986). Additionally, the transcriptional trans-
activator Tat was found to affect elongation rather than
initiation during its regulation of HIV transcription (Kao
et al., 1987). In another study, it was found that RNAPII
is paused close to the HSP70 promoter at normal tem-
peratures in D. melanogaster (Rougvie and Lis, 1988).
Events promoted by temperature shift then release
RNAPII to synthesize full-length HSP70 transcripts
(Rougvie and Lis, 1990). Over the past two decades, it
has become clear that what happens on the c-myc
and HSP70 genes as well as on the HIV long terminal re-
peat (LTR) is more the rule than the exception. Indeed,
reversing an early block to RNAPII elongation is how
many genes are regulated in organisms from flies to hu-
mans (Chao and Price, 2001; Price, 2000).
A significant advance in our understanding of this
process came from studies utilizing the ATP analog
They demonstrated that DRB leads to a dramatic reduc-
tion in mRNAlevels and the appearance ofshort capped
transcripts (Sehgal et al., 1976). Importantly, DRB in-
hibited transcription elongation in vitro (Chodosh et al.,
1989). Subsequently, studies using a fly transcription
system led to the formulation of a model for controlling
the movement of RNAPII (Marshall and Price, 1992) (Fig-
ure 1). It states that after initiation, RNAPII comes under
the control of negative transcription elongation factors
(N-TEF) and the elongation complex is trapped near
the promoter. However, P-TEFb overcomes the influ-
tive elongation. Indeed, P-TEFb rather than RNAPII is
the target of DRB (Marshall and Price, 1995).
Over the past decade, the identity of P-TEFb was re-
vealed. It is a kinase that phosphorylates the C-terminal
domain (CTD) of the large subunit (RPB1) of RNAPII
(Marshall et al., 1996). Subsequent cloning of the two
subunits of P-TEFb from flies and humans revealed it
as PITALRE (Grana et al., 1994), because it requires a
C type cyclin subunit, it was later renamed Cdk9. Flies
express cyclins T1 and T2 (CycT1 and CycT2) (Peng
et al., 1998b). The human Cdk9 protein also binds cyclin
K (CycK) (Fu et al., 1999).
Two negative factors with properties consistent with
N-TEF were also identified. First, the DRB-sensitivity in-
ducing factor (DSIF) is required for the effects of DRB
and P-TEFb on transcription in vitro (Wada et al.,
et al., 1999b). It contains two subunits, which are similar
to yeast transcription factors Spt4 and Spt5 (Winston,
2001). Subsequently, the negative elongation factor
(NELF) was found to be necessary for the negative func-
tion of DSIF (Yamaguchi et al., 1999a). Following their
characterization, recombinant DSIF and affinity-purified
NELF were used in a defined in vitro transcription sys-
tem along with recombinant P-TEFb to recapitulate
essential features of elongation control (Renner et al.,
2001). Although neither factor had any effect alone, the
combination of DSIF and NELF slowed the elongation
of RNAPII and this effect was eliminated by P-TEFb. In
a similar in vitro system, NELF and DSIF also inhibited
the transcript cleavage factor TFIIS (Palangat et al.,
2005). Because the function of TFIIS is to relieve strong
pauses and arrests, this inhibitory property could stabi-
lize the paused RNAPII conformation induced by NELF
andDSIF. Thefunction ofNELF andDSIFwasconfirmed
by studies that demonstrated that these two factors in-
duce promoter proximal pausing on the HSP70 gene
from D. melanogaster in vitro (Wu et al., 2003).
What are the targets of P-TEFb? Many studies indi-
cate that P-TEFb phosphorylates the C-terminal domain
(CTD) of RNAPII primarily on serines at position 2 (serine
2) of its heptapeptide (YSPTSPS) repeats (Garriga and
Grana, 2004; Price, 2000). The phosphorylation of the
CTD, which in humans contains 52 such repeats, is a
complicated process. At least two different serine/thre-
onine kinases are required (Figure 1). First, Cdk7 from
*Correspondence: email@example.com (B.M.P.); david-price@
TFIIH, which is a general transcription factor, phosphor-
ylates serines at position 5 (serine 5). Next, Cdk9 phos-
phorylates serine 2 (Figure 1). This posttranslational
modification increases the diameter and rigidity of the
CTD. In the process, most proteins in the preinitation
complex, the Mediator, and general transcription fac-
tors, which comprise 100 or more proteins, are removed
from RNAPII. The phosphorylated CTD now increases
the affinity of human capping enzymes (HCEs) for the
elongation complex and acts as a scaffold for splicing
(SR) and polyadenylation (pA) machineries (Figure 1).
In addition, chromatin-modifying enzymes and other
elongation factors also travel with the phosphorylated
RNAPII. As presented in Figure 1, these steps are se-
quential and have been subdivided into the formation
of the preinitiation complex (PIC), promoter clearance,
50capping, and pausing followed by productive elonga-
tion(Simsetal., 2004). ThelaststeprequiresP-TEFb.In-
deed, when Cdk9 or its two cyclin T subunits are genet-
ically inactivated in C. elegans, serine 2 phosphorylation
is lost (Shim et al., 2002). The development of these
to that observed when a subunit of RNAPII is knocked
tein (Kim and Sharp, 2001). Thus modified, DSIF func-
tions as a positive elongation factor (Yamada et al.,
2006). Finally, P-TEFb phosphorylates NELFe (RD).
This phosphorylation releases NELF from the double-
stranded RNA and leads to some read through tran-
scription (Fujinaga et al., 2004).
Recruitment of P-TEFb
For its effects on RNAPII, P-TEFb must be recruited to
transcription units. Some specific activators are known
torecruit P-TEFbas well asatleast one general chroma-
tin remodeling protein, Brd4 (Jang et al., 2005; Yang
et al., 2005) (Figure 2). However, certain specific or gen-
eral repressors, such as PIE-1 from C. elegans, can
block these effects (Figure 3), keeping RNAPII arrested
despite the presence of P-TEFb (Zhang et al., 2003).
Figure 1. Steps of Transcription by RNAPII
Four stages of transcription are depicted: (1)
preinitiation complex (PIC) formation, (2) pro-
moter clearance with the phoshorylation of
serines at position 5 of the CTD, (3) recruit-
ment of human capping enzymes (HCE), 50
capping (m7G) of nascent transcripts, and
pausing of RNAPII, and (4) recruitment of
P-TEFb with the phosphorylation of serines
at position 2 in the CTD, followed by produc-
tive elongation and cotranscriptional pro-
cessing by splicing (SR) and polyadenylation
(pA) machineries. Phosphorylated serines at
positions 2 and 5 in the heptapeptide repeats
of the CTD of the large subunit of RNAPII are
depicted by white letters in red circles.
Figure 2. Recruitment of P-TEFb to Tran-
Depicted are five mechanisms responsible
for the recruitment of P-TEFb to genes: (1) ar-
tificial tethering (Gal4 DNA binding domain
fusions), (2) coactivator (CIITA), (3) DNA
bound activator (NF-kB, c-Myc, and nuclear
receptors), (4) RNA bound activator (HIV
Tat), and (5) chromatin bound activator
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