PROMOTER-ASSOCIATED PAUSING IN PROMOTER ARCHITECTURE AND

POST-INITIATION TRANSCRIPTIONAL REGULATION

John Lis. Section of Biochemistry, Molecular and Cell Biology, Biotechnology Building,

Cornell University, Ithaca, NY 14853.

Eukaryotic Transcription Provides a Rich Repertoire of Regulatory Targets:

Multiple Factors and Discrete Steps.

The transcriptional regulation of a eukaryotic gene is specified by the interplay of specific

regulatory factors, general transcription factors (GTFs), RNA polymerase II (Pol II), DNA

sequence elements, and the chromatin structure of the promoter. The thousands of genes in a

eukaryotic cell share a general transcriptional machinery and a common transcriptional pathway

(Orphanides et al. 1996). Nonetheless, variations in regulatory mechanisms are becoming

increasingly apparent.

Hundreds of specific upstream activators and repressors have been identified. These

proteins bind near a particular target gene either through specific interactions with DNA sequences

or by specific protein interactions with other proteins that are themselves targeted to specific DNA

sequences. The combination of factors that can interact with a specific gene collaborate with the

GTFs and the core promoter to set the regulatory mechanisms of the gene. The copious number

and combinations of specific factors provide for a rich repertoire of regulation. In addition to this

variety of upstream factors, the GTFs also show variety in that variant GTFs or alternative

complexes containing GTFs add additional sophistication to the regulatory process (Grant et al.

1997; Hansen et al. 1997).

The regulation of mRNA-encoding genes generally occurs at steps early in the transcription

cycle and can be specified by core promoter DNA (-40 to +40), which contains the TATA box,

the transcription start, and the downstream promoter element (Burke and Kadonaga 1997), and a

few hundred base pairs of upstream sequences, which contain gene specific activator and repressor

binding sites. This expression can be further influenced in some genes by enhancer regions that

can reside thousands of base pairs from a transcriptional start site. Fusing the RNA leader of a

particular gene (c.a. +40) to a reporter gene is often sufficient to recapitulate the expression level

and regulatory properties of the normal gene. This indicates that transcription beyond about +45 of

the transcription unit is usually not a critical part of the regulation. Nonetheless, for Pol II to

progress to this point and form a competent elongational complex requires numerous distinct steps

and a large battery of interacting general factors. Any of these steps and general factors can

potentially be rate limiting and a target of regulation by specific activators and repressors.

Some of the prominent steps in producing an elongationally competent Pol II complex are

outlined in Figure 1.

1. Opening chromatin . Chromatin structure can create an early barrier to gene expression and in

some cases can prevent access of transcription factors to the promoter (Taylor et al. 1991). The

ability of GTFs to gain access to their target sequence is likely to be facilitated not only by

sequence-specific DNA binding factors, but also by chromatin remodeling machines (Wu 1997).

2. Binding TFIID. A critical foundation of the promoter is the GTF complex that contains TBP

and can interact with the TATA box or start site region or both (Purnell et al. 1994).

3. Recruiting the pre-initiation complex. Other factors and Pol II are then recruited via ordered

assembly (Buratowski et al. 1989) or as a holoenzyme that contains many of the GTFs, mediator,

and the core Pol II (Thompson et al. 1993; Kim et al. 1994).

4. Forming the open complex. The bound Pol II progresses from a closed to an open complex

(Holstege et al. 1997).

5. Initiation. Pol II forms the first phosphodiester bond to initiate transcription. Pol II at this

stage can synthesize and release short 7-14 nucleotide transcripts reiteratively (Holstege et al.

1997).

6. Promoter clearance. Pol II passes beyond this phase of synthesizing short abortive transcripts

as it acquires a longer, stably-associated RNA (Goodrich and Tjian 1994).

7. Promoter escape. Pol II with relatively short, stably associated RNAs of 18 to 45 nucleotides

can pause. The production of a fully-competent elongational complex is defined as escape.

A2

Pol II

TATA

1. Opening Chromatin

B

RNA

A1TATA

2. Binding TBP

F

Med

3. Recruiting Pol II

4. Open Complex

5. Initiation

6. Clearance

7. Escape

NTPs

EH

TBP

Figure 1. Steps in early transcription. Each step is described in the text. The labels identifyvarious factors and complexes: TBP is TATA-binding protein; Med is the mediator complex ofthe Pol II holoenzyme; F, E, H, and B are the corresponding TFII general transcription factors;and Pol II is RNA polymerase II. A1 and A2 represent specific activators shown here to beacting early and late in the process, though in principle they could act at any regulated step.

The steps outlined in Figure 1 provide a relatively low resolution view of mechanism.

Each of these steps may be further divided. For example, the recruiting of a pre-initiation complex

can occur via one (Thompson et al. 1993). or multiple distinct steps (Buratowski et al. 1989).

When considering molecular mechanisms of activator or repressor function, the step or steps

affected may need to be considered at these higher levels of molecular detail.

A Case For Regulation At The Level Of Promoter Escape: Drosophila Hsp70.

The Drosophila Hsp70 gene is rapidly and vigorously activated by heat shock. An

instantaneous heat shock triggers a 200-fold increase in the level of transcription in three minutes

(O'Brien and Lis 1993). This activation is orchestrated by the DNA sequence-specific activator,

heat shock factor (HSF) which upon heat shock rapidly trimerizes and binds to heat shock loci

(Westwood et al. 1991). The framework for this rapid activation was first suggested by

measurements demonstrating the DNAaseI hypersensitive structure of the hsp70 promoter (Wu

1980). This open chromatin configuration could provide HSF and the general transcription

machinery the rapid access to specific sequences of the promoter of heat shock genes. This

promoter appears to be further primed for transcription in that Pol II and TBP are already an

integral part of the hsp70 promoter even before heat shock activation.

Four distinct classes (I-IV) of measurements performed directly on Drosophila cells or

intact nuclei support the existence of Pol II on 5 ' end of uninduced hsp70 genes (Fig. 2). These

assays quantify the amount of this polymerase, delimit its precise location , and define some of its

features. Approximately one Pol II is transcriptionally engaged, but paused, on each hsp70 gene

(Rougvie and Lis 1988). This Pol II is paused at sites covering the interval from +21 to +35, with

two peaks of pausing within this interval that are separated by a turn of the DNA helix (Rasmussen

and Lis 1993). This Pol II is largely hypo-phosphorylated (O'Brien et al. 1994), and its associated

short RNA is uncapped when Pol II is at the start of the paused interval but is largely capped on the

distal portion on pause region (Rasmussen and Lis 1993).

Hsp70

-12 +65

+1

I. UV-Crosslinking:One Pol II per hsp70 gene.

II. Nuclear run-on:

III. KMnO 4 mapping:Transcription bubble.

IV. Sizing paused RNA:Pausing region.

Pol II

N. DNA

NHS

HSCel

ls

Pol

II

Nucleotide

+3 +8 +22 +30 +40

1NHS

HSPol

II

10.1530de

nsity

+2440

+20 +40

0.1

0.05

Figure 2. Summary of four different classes of experiments describing the paused Pol II on the Drosophila hsp70 gene. I) UV crosslinking andimmunoprecipitation analyses first revealed the high density of Pol II on the 5' end relative to the body of the hsp70 gene in uninduced (NHS)(Gilmour and Lis 1986). The corresponding levels in induced (HS) cells were also derived from these experiments and are illustrated below thehsp70 map. II) Nuclear run-on assays demonstrated that the density of transcriptionally-engaged Pol II is virtually identical to that seen bycrosslinking (Rougvie and Lis 1988). III) Potassium permanganate treatments of intact cells identified sites on the hsp70 gene that are hyper-reactive relative to that seen in naked DNA (N.DNA). These cover the region of the expected transcription bubble created by the pausedpolymerase (Giardina et al. 1992). IV) Distribution of pause sites was determined on the uninduced hsp70 by sizing RNAs associated with thepaused polymerase (Rasmussen and Lis 1993). The graph represents relative densities (normalized to a total area of 1 paused Pol II) at differentpositions in the pause region.

I. Crosslinking. The initial evidence for Pol II association with the uninduced hsp70 gene

was obtained over a decade ago from in vivo crosslinking and immunoprecipitation (Gilmour and

Lis 1985;Gilmour and Lis 1986). When developing our UV-crosslinking approaches to measure

protein density on specific DNA sequence in vivo, we were surprised to find a high density of Pol

II on the 5' end of the uninduced Drosophila Hsp70 gene, because it had been assumed that all

transcription was regulated at the level of Pol II recruitment or "initiation". We had expected that

a low level of polymerase would exist on the gene prior to heat shock and the level would increase

200-fold upon activation, mirroring the change in transcription as seen in the analysis of pulse-

labeled transcripts (Lis et al. 1981). While the 3' half of the gene showed the expected increase in

polymerase density, the promoter region (-12 to +65) had a density of polymerase before heat

shock that was equivalent to one Pol II per gene. This absolute estimate was calculated from the

crosslinking of polymerase to the heat-shock-induced gene, where rates of RNA synthesis and

direct EM visualization of growing RNA chains in Miller spreads indicate 30 RNA polymerases are

on the fully-activated hsp70 gene (O'Brien and Lis 1993). The Pol II seen by UV crosslinking

was shown not to be simply an artifact of recruiting Pol II during the UV irradiation of cells, since

the same density is observed with a 10 min. irradiation with mercury lamps as is seen by a 60 _sec

flash with a xenon flash lamp (Gilmour and Lis 1986). More recently, we have used UV

crosslinking and antibodies specific to the hypo- or hyper-phosphorylated Pol II to show that the

paused Pol II is hypo-phosphorylated (O'Brien, et al. 1994).

II. Nuclear run-on. A Pol II that crosslinks to the 5' end of hsp70 gene could in principle

be at any of a number of discrete steps in the process of early transcription (Fig. 1). Nuclear run-

on assays demonstrated that this Pol II has initiated transcription but is elongationally paused. In

isolated nuclei from uninduced cells, the promoter-associated Pol II is capable of transcribing

under conditions that prevent transcription initiation. A short run-on reaction performed in the

presence of sarkosyl or high salt concentrations shows a large burst of transcription that is

restricted to the 5' end of the gene (Rougvie and Lis 1988), and comparison of the amount of this

run-on product to that from induced nuclei indicated that there is one engaged Pol II per hsp70

gene. Longer run-on reactions demonstrate that this polymerase can progress through the body of

the gene. Interestingly, if nuclei are very carefully prepared from uninduced cells and no sarkosyl

or high salt is added, this promoter-associated polymerase transcribes very inefficiently in a run-on

reaction. From these results, we hypothesized that a transcriptionally-engaged Pol II resides on the

5' end of the hsp70 gene and that it is normally paused in vivo (Rougvie and Lis 1988).

III. Permanganate "Bubble Mapping". The evidence that the promoter-associated Pol II is

in a paused configuration was derived initially from analysis of run-on reactions performed with

isolated nuclei. To examine cells directly, we (Giardina et al. 1992) used the reagent potassium

permanganate which preferentially modifies T residues in single-stranded DNA. We reasoned that

a single-stranded DNA bubble should lie in the wake of a paused Pol II and be detected by

permanganate. After a brief 30" permanganate treatment of cells, DNA was purified and sites of

modification cleaved. Ligation-mediated PCR revealed the sites of modification and mapped the

single-stranded region to an interval consistent with that expected from the location of pause sites

mapped by sizing RNAs (see below and Fig. 2). Thus, analysis of intact Drosophila cells with a

brief (30") chemical treatment provides evidence for pausing in vivo.

IV. Sizing paused RNAs. The precise location of the pause or pauses could be determined

by sizing the short, rare RNAs associated with the paused polymerase. To achieve this, we

developed new sensitive approaches for purifying and assaying rare RNAs. In the first strategy

(Rasmussen and Lis 1993) radiolabeled, chain-terminated RNAs were generated by performing

run-on reactions in the presence of various combinations of radiolabeled nucleoside triphosphates

and chain terminating nucleoside triphosphates. The short RNAs were hybridized to completion

with excess biotin-labeled oligonucleotide, and the biotin oligonucleotide complexes were

recovered with avidin-coated magnetic beads. After extensive washing, the labeled RNAs were

sized by gel electrophoresis, and the pause sites deduced from the sizes obtained with different

combinations of labeled and chain-terminating NTPs. The pause sites reside between position +21

and +35 with two peaks of pausing separated by approximately one turn of the DNA helix. This

suggests a sidedness to pausing where the Pol II may interact with factors that inhibit its progress;

however, other explanations are possible. Interestingly, the isolated RNAs contain a mixture of

capped and uncapped species (Rasmussen and Lis 1993). RNAs near the start of the pause region

are largely uncapped, while those at the end are fully capped. This unexpected, extra information

from these assays define the point during RNA synthesis where capping occurs in vivo and this

result agrees well with in vitro studies of capping with vacinia virus which also show that capping

occurs early in transcription (Hagler and Shuman 1992).

The second strategy for sizing paused RNAs again made use of biotin oligonucleotides to

select unlabeled RNAs extracted from nuclei. These RNAs were then amplified and labeled by

ligation-mediated PCR (Rasmussen and Lis 1995). Analysis of the size of amplified fragments

allowed the derivation of pause sites. These results are in excellent agreement with the first

strategy, and this approach is more sensitive and could be applied to genes that have relatively low

levels of paused Pol II (Rasmussen and Lis 1995).

TBP and GAGA Factor (GAF) Also Occupy the Uninduced Hsp70 Promoter.

A byproduct of transcription bubble mapping with permanganate was the ability to detect

TBP protection of the TATA sequences. TATA sequences are generally in non-B-form DNA and

are hypersensitive to modification with potassium permanganate. In cells, however, the hsp70

(and hsp26) TATA sequences are relatively protected from permanganate modification (Giardina,

et al. 1992). Since this pattern of protection appears identical to that generated with purified cloned

promoter DNA and purified recombinant TBP, we conclude that TBP is bound to the TATA box of

the uninduced hsp70 promoter.

GAF appears to have an important role in heat shock gene expression. Mutations in the

strong binding sites (GA repeats) for GAF impair the function of the hsp70 and hsp26 promoters.

GAF is not a traditional transcription activator in vitro, but appears to have a role in overcoming

repression imposed by histones (Wilkins and Lis 1997). Since GAF is constitutively present in

nuclei and is a prime candidate for having a role in establishing the potentiated promoter, we

examined GAF's occupancy of various promoters in cells directly by UV crosslinking and

immunoprecipitation (O'Brien et al. 1995). These studies demonstrate that GAF is present on the

uninduced hsp70 and hsp26 promoter regions.

The resulting image of the architecture of the uninduced hsp70 promoter is depicted in

Figure 3. An open chromatin structure extends over all critical features of the promoter. Within

this region are a single paused Pol II that is distributed over the region +21 to +35, GAF

interacting with the GAGA sequences, and TBP occupying the TATA box. Together, these

various features of the hsp70 promoter constitute what we have called the potentiated promoter (Lis

and Wu 1995). Additional GTFs could also be present at the potentiated promoter, and additional

crosslinking and immunofluorescence experiments are required to determined which are present,

where precisely they are located, and how their distribution changes upon activation.

DNA Sequences Critical in Establishing and Activating the Potentiated Promoter.

The functional elements of the hsp70 promoter have been identified by analysis of

transgenic fly lines containing a variety of promoter mutations. Initially, these analyses

demonstrated the critical role of heat shock elements (the targets to which HSF bind) for heat-

induced activation (Xiao and Lis 1988). While mutations in the elements to which HSF bind

affect heat shock gene activation, they have little effect on establishing the paused Pol II (Lee et al.

1992). The full activation of heat shock promoter was also found to be dependent on GAGA

elements, the targets to which GAF bind (Glaser et al. 1990;Lee et al. 1992). In addition, these

GAGA elements were found to be critical for the formation of the nuclease hypersensitivity of the

hsp26 and hsp70 promoters (Lu et al. 1992;Shopland et al. 1995). Interestingly, the GAGA

elements were also found to be important for establishing a paused Pol II and bound TBP

(Shopland et al. 1995). Therefore, GAF protein and GAGA elements are critical for generating the

potentiated promoter. Full heat-induced activation also requires GAF either to form this promoter

structure or to participate directly in the activation.

Sequences of the core promoter region were also found to be required for generating

paused Pol II (Lee et al. 1992). While deletions of the regions downstream of +30 cause only a

Heat Shock

Nucleosome

GAGA FactorPol II

HSF

RNA

Hsp70

CTD

TBP

1. HSF-Inducedmodification of Pol II

2. Competitive bindingof HSF and Pol II to corefactors

3. Termination andinitiation ofactive Pol II

3 Activation Models:

Figure 3. Model depicting the hsp70 promoter architecture before and after heat shock activation. Theminus signs associated with the CTD (C-terminal domain of the largest subunit of Pol II) indicate the hyper-phosphorylated state. The three models of activation and other features of this sketch are described in thetext.

modest (1.5 fold) reduction in paused Pol II and a 2-fold reduction on the activation of

transcription following heat shock, a 3' deletion that extends to position +23 results in a greater

reduction in the level of paused polymerase on the hsp70 gene (3.5 fold) and also further impairs

its inducibility in response to heat shock. Deletions entering farther into the core promoter show

even greater reduction in pausing (to background levels) and further reduce heat-inducible

transcription. Therefore, both GAGA sequences and core promoter sequences extending through

the pause region are important in establishing the potentiated hsp70 promoter and its subsequent

activation.

Interestingly, although the mutagenesis of the hsp70 promoter is far from exhaustive, none

of the mutations in the hsp70 promoter lead to an elevated constitutive expression of hsp70. Such

mutations might be expected if specific sequences elements bound factors that acted as blockades to

Pol II. Also, no mutation has been found that exhibits normal pausing and disrupts the

inducibility of hsp70. In contrast, we find that the mutations that reduce pausing, also reduce the

inducibility of the gene. Thus, the degree of pausing and the strength of the activated promoter

appear to be coupled.

Why Build a Potentiated Promoter? Speed and Access.

Stress rapidly induces genes that have paused Pol II (Stewart et al. 1990; O'Brien and Lis

1993) and it is tempting to speculate that the open promoter with a transcriptionally-paused Pol II

may provide such a gene with the ability to be very rapidly activated. The first wave of

polymerase is already engaged in transcription before induction, and the recruitment of additional

Pol II may be facilitated by this promoter architecture that is open and includes pre-bound TBP.

Kinetic analyses using UV-flash-crosslinking and nuclear run-on assays directly revealed the

dynamics of Pol II movement and accumulation on the hsp70 gene in Drosophila cells (O'Brien

and Lis 1993). The first wave of Pol II moves detectably beyond the pause region of the hsp70

gene within 70 seconds following an instantaneous heat shock. Within 3 minutes the density of

Pol II on hsp70 is near its fully induced level. This rapid activation appears similar to that

observed for c-fos (Stewart et al. 1990) which also has a promoter-paused Pol II [(Plet et al. 1995)

and see below].

The open structure of the heat shock promoters is also critical for the rapid recruitment of

HSF. In vitro, Kingston and colleagues (Taylor et al. 1991) have shown that HSF binds much

less well to heat shock elements that are in nucleosomes than in naked DNA. Transgenic lines that

carry hsp70 gene promoter mutations provided an opportunity to assess the effects of promoter

architecture on HSF binding in vivo. Immunofluorescence assays of Drosophila polytene

chromosomes can be used to localize sites of HSF binding. Indeed, transgenic lines carrying

functional hsp70 genes create new bands of HSF at the sites of insertions (Shopland et al. 1995).

Surprisingly, mutations that disrupt the leader sequences underlying the paused polymerase, but do

not disrupt heat shock elements, reduce (to undetectable levels) the heat-shock-induced recruitment

of HSF to the mutant transgene as seen by immunofluorescence and footprinting (Shopland et al.

1995). These mutants prevent the formation of paused polymerase and alter the architecture of the

uninduced promoter. Likewise mutations in the major GAGA element of the hsp70 transgenes

also prevent pausing and reduce the induced HSF binding to this transgene (Shopland et al. 1995).

Binding of TBP to the hsp70 promoter in vivo is also affected in both of these classes of mutation.

Therefore, it appears that the overall promoter architecture is dependent upon the interplay of the

TBP, GAF, and the paused polymerase, and that this in turn determines whether the heat shock

promoter is accessible to HSF.

Mechanism of Transcription Activation: Pol II Recruitment Is Not Sufficient.

Heat shock triggers the trimerization and highly specific binding of HSF to chromosomal

sites (Westwood et al. 1991). While many chromosomal sites recruit HSF, the most prominent

sites of HSF localization are the loci containing heat shock genes. HSF binds tightly to the

multiple heat shock elements of these genes and triggers the 200-fold increase in transcription of

major heat shock genes. To account for the basal level of hsp70 expression in uninduced cells, Pol

II must escape the pause mode and enter into productive elongation once every 10 minutes. After

heat shock and HSF binding to the promoter, Pol II must escape to productive elongation once

every 4 seconds (this follows from the density of polymerase being one Pol II per 80 base pairs

and the elongation rate being 1.2 kb/min. (O'Brien and Lis 1993)). The mechanism by which

HSF stimulates this dramatic and rapid increase in transcription is not understood, but at least three

models should be considered. While these models differ in detail, they all have the common

feature of being clearly distinct from regulatory models where activators simply recruit Pol II to the

promoter directly or by recruiting GTFs that in turn recruit Pol II (Ptashne and Gann 1997). Pol II

is already recruited to promoters that display pausing, and this recruited Pol II encounters what

appears to be a rate-limiting step early in its elongation.

1) Modification of the paused Pol II complex. Paused Pol II is impaired in its elongational

competence relative to polymerases that have progressed beyond the pause region. Perhaps the

paused Pol II is modified to an elongationally competent form in response to HSF activation. A

modification could alter Pol II's properties or associations, allowing it to progress to an

elongationally competent form. The modification could potentially be of a component of this

paused structure other than Pol II itself, such as chromatin structure, since studies by Kingston's

group of pausing on human hsp70 support a role of chromatin modification in HSF activation

(Brown and Kingston 1997). In contrast, studies by Gilmour and colleagues of Drosophila hsp70

promoter pausing in vitro show that the pause can form in nuclear extracts in the absence of

assembled chromatin (Li et al. 1996) and even on templates too short to support a downstream

nucleosome (Benjamin and Gilmour 1998). Perhaps the resolution of these apparently

contradictory conclusions is that the pause can be specified in the absence of chromatin, but the

level of pausing is enhanced (and regulated) by chromatin and the degree of this enhancement may

vary for different genes or hosts.

One modification of Pol II that strongly influences its properties is the phosphorylation of

the C-terminal domain (CTD) of the largest subunit. The form of Pol II that can enter a promoter is

hypo-phosphorylated, while the elongational form is hyper-phosphorylated (Lu et al. 1991;

Dahmus 1994).

Greenleaf and colleagues (Weeks et al. 1993) examined the chromosomal distribution of

hypo-phosphorylated and hyper-phosphorylated epitopes of the Pol II CTD by

immunofluorescence of polytene chromosomes. Many chromosomal sites are sharply labeled by

antibody to the hypo-phosphorylated form of Pol II , while the hyper-phosphorylated form of Pol

II is associated with numerous diffuse puffs and interbands many of which show little overlap with

sites of the hypo-phosphorylated polymerase. Sites containing inserted Drosophila hsp70

transgenes(Lis et al. 1983) show new (relative to wild type) sharp bands of hypo-phosphorylated

Pol II in uninduced animals (Weeks, et al. 1993). Presumably, these are the paused polymerases

associated with the hsp70 genes. Upon heat shock, the large puffs generated at these sites are

labeled with antibodies to both forms of Pol II.

At higher resolution, O'Brien et al. (O'Brien, et al. 1994) examined the distribution of the

different forms of Pol II on several genes using UV crosslinking and immunoprecipitation. These

studies show that the paused Pol II at the start of uninduced heat shock genes is indeed hypo-

phosphorylated, while the Pol II population on the body of the induced gene is composed of

polymerases that contain both the hyper- and hypo-phosphorylated epitopes. The transcribing

polymerases contain some heptapepide repeats of the CTD that are and some that are not

phosphorylated. Therefore, the paused Pol II lacks phosphorylation and is like the form that enters

the promoter (Dahmus 1994). In contrast, the activated body of the gene is covered with

phosphorylated Pol II.

It is tempting to consider that the escape of paused Pol II may depend on a phosphorylation

by a kinase that is either recruited or activated by HSF. The general factor TFIIH is composed of

multiple subunits that include an essential helicase activity and an essential cyclin dependent kinase

(CDK7/KIN28), which appears responsible for a significant portion of phosphorylated Pol II CTD

in yeast (Cismowski et al. 1995;Valay et al. 1995). Indeed, TFIIH is recruited to Drosophila heat

shock loci during heat shock, however, it is not clear if this is a requirement for the essential

helicase or kinase activities or both (Schwartz and Lis unpubl. results). TFIIH kinase becomes

largely insoluble in extracts upon (maximal) heat shock temperature treatments of HeLa cells

(Dubois et al. 1997), indicating that heat shock promoters may be built in ways that make use of

specialized conditions or requirements of heat shock. These findings leave open the possible

participation of another kinase that may be recruited or stimulated by HSF.

2) Competition of HSF with Pol II for the core promoter. Promoter-associated pausing may be a

consequence of Pol II's affinity for a strong core promoter. Mutations in the core promoter reduce

both pausing and heat-induced activation on hsp70 (Lee, et al. 1992). The heat shock promoters

are extremely strong, presumably a consequence of their open chromatin configuration and their

extensive core promoter-TFIID interactions (Purnell et al. 1994), and they allow for rapid Pol II

entry. Some of the same interactions that assist Pol II binding to the promoter may persist and

slow its escape. Pol II may be able to initiate and begin transcription, but it is tethered and lacks

the ability to break away into a fully elongational mode.

Perhaps the binding of HSF disrupts Pol II/core promoter interactions by direct competition

with RNA Pol II for binding to components of the core promoter. In this regard, it is of interest

that HSF is an acidic activator that binds very tightly to TBP (Mason and Lis 1997) and that Pol II

itself possesses a strong acidic activation domain in the H-homology region (adjacent to the CTD)

of the largest subunit of Pol II (Xiao et al. 1994). This region can bind competitively with HSF

for a region on TBP, and a single point mutation in TBP (L114K) disrupts binding to TBP of both

HSF and the H region of Pol II (Mason and Lis 1997). We do not know if the H-region of Pol II

contacts TBP in vivo. The possibility of such an interaction is not inconsistent with the intriguing

observation that mutations in the critical phenylalanines of the H-domain of the largest subunit of

yeast RNA Pol II influence transcription start site selection in vivo, allowing Pol II to reach start

sites farther downstream (H. Xiao and E. Guzman, unpublished results).

3) HSF-induced replacement of paused polymerase with different elongationally-competent

polymerases. One question that is extremely difficult to resolve unambiguously is whether the

paused polymerase II is actually the polymerase that enters into productive elongation. Models

have been proposed that suggest that the upstream activators of genes act to trigger new Pol II

initiations that, through the participation of upstream activators, are elongationally competent and

displace the paused Pol II which is elongationally incompetent (Cullen 1993;Krumm et al. 1993).

In such a model the paused polymerases may have a role in maintaining an open promoter but do

not contribute directly to the population of elongating Pol II. The main problem with this class of

model comes from a quantitative consideration of the levels of pausing on the active gene and its

implications concerning the mechanics of the process.

The fact that pausing persists on the activated hsp70 gene makes this third model harder to

rationalize. The hsp70 paused polymerase is evident in run-on assays in cells induced at sub-

maximal heat shock temperatures that produce a low enough density of transcribing polymerase to

allow detection of the paused polymerase (O'Brien and Lis 1991). With a fully-induced hsp70

gene, we have also demonstrated with the high resolution potassium permanganate assay that

pausing occurs at nearly the same steady state level as seen in uninduced cells (Fig. 2 and Giardina

et al. 1992). Since the level of pause region occupancy is one Pol II/gene, a fully induced gene

that fires an elongationally-competent RNA Pol II every four seconds must instantly reestablish a

paused Pol II. This new paused polymerase would remain for four seconds only to be displaced

by a newly initiated and elongationally competent Pol II. The process would then have to be

repeated with each round of transcription to account for the steady state occupancy of the pause site

during heat shock. This seems mechanistically clumsy and improbable, though it is difficult to rule

out. This model is easier to accept if the elongationally competent polymerases can also encounter

the slow pausing step of early elongation, but because the Pol IIs have been modified, they spend

only four seconds rather than 10 minutes at this rate-limiting step.

Resolving the fate of the paused polymerase is technically difficult, and to date, the

attempts have not been completely satisfying. On one hand, the paused Pol II is engaged in

transcription, and in nuclear run-on assays, is capable of elongating deep into the body of the

hsp70 gene (Rougvie and Lis 1988). These results demonstrate that the paused polymerase can

transcribe. Moreover, a fraction of the paused polymerase is associated with RNAs that are capped

and appear ready for elongation (Rasmussen and Lis 1995). On the other hand, in an attempt to

examine transcripts after a one minute instantaneous heat shock, we have observed that a

significant fraction of short transcripts were no longer chased to longer transcript when run-on

reactions were done in the presence of sarkosyl (Rasmussen and Lis 1995). We used the short-

hand of referring to these as terminated transcripts in that paper. If indeed they are truly

terminated, then under these conditions some paused polymerases do not give rise to elongating

complexes. These experiments however do not distinguish between terminated RNAs and RNAs

associated with arrested polymerases. It is possible that at least some paused Pol II molecules

progress to an intermediate state (indeed most of the transcripts that become non-elongateable are

of a length between the two peaks of normally paused RNAs) that is not capable of transcribing

and only upon full modification can they escape to productive elongation. Additionally, these

experiments need to be re-examined in a manner that provides a more natural heat shock protocol

where the temperature is not raised instantaneously. Finally, this particular assay, while very

sensitive, needs to be developed into a quantitative assay, to allow a strict accounting of RNAs that

are paused, arrested, elongating, and terminated.

The three models described are not mutually exclusive. First, Pol II may require more than

one molecular event to escape the pause, the modification (model 1) and competition (model 2)

could both participate as means of triggering Pol II escape. Second, the modification of

polymerase during activation in model 1 may occur not only late in the process, when Pol II is

paused, but also at various early steps in transcription as in model 3. This modification whether

early or late could reduce the length of time it takes for Pol II to escape the pause region.

Promoter-associated Pausing is Somewhat General: c-myc, c-fos and others.

The heat shock gene promoters and regulatory regions appear to use much of the same

transcriptional machinery as other genes. The upstream and core promoter elements of hsp70 and

the associated protein factors can function with elements and factors of other genes (Lis and Wu

1995). Enhancers of developmentally regulated genes can drive expression of an hsp70 promoter,

and heat shock elements can often drive the expression of other core promoters. These hybrid

combinations may not always work as efficiently as native Hsp70, but at some level the regulatory

and core machinery of a variety of genes can communicate.

While this article has as its focus the pausing on major heat shock genes, a variety of other

Drosophila genes, such as ß1-tubulin and Gapdh-1 and -2 show some level of paused Pol II.

These genes display higher densities of Pol II at their 5' ends than in the body of the gene as

judged by in vivo UV crosslinking/immunoprecipitation experiments, and this extra 5' Pol II (over

the density found on the body of the gene), like the paused Pol II of heat shock genes, is

stimulated to transcribe in the presence of sarkosyl or high salt (Rougvie and Lis 1990). The levels

of pausing on other genes is not as high as on the hsp70 gene, where we estimate one Pol II

molecule per promoter. These constitutively-expressed genes presumably load and fire paused Pol

II at rates that lead to a lower steady state level than seen for the hsp70 gene. Alternatively,

perhaps these constitutive genes are governed by a stochastic process such that only a fraction of

the cells have active genes and paused Pol II.

Immunofluorescence studies of Greenleaf and colleagues using antibody to hypo-

phosphorylated CTD of Pol II of polytene chromosomes showed that there are many sites (over

100) that are labeled (Weeks et al. 1993). The hsp70 loci and new hsp70 loci in transgenic lines

contain the hypo-phosphorylated form of Pol II. It is tempting to speculate that these other sites

also represent paused polymerases like those seen on hsp70. The test of this idea requires

additional experimentation.

Elongational control of c-myc has been known for some time. While initial in vitro and

Xenopus injection studies localized the regulation at first exon/intron border in the region

surrounding +400, further analysis of nuclei with run-on assays and in cells with potassium

permanganate localized the block to c-myc elongation to a promoter pause site at an interval

centered at +30 (Krumm et al. 1992;Strobl and Eick 1992). This paused Pol II is remarkably

similar to that on Drosophila hsp70 in terms of its location and properties (Krumm et al 1995;

Albert et al. 1997). The c-fos gene also appears to have paused Pol II that is very similar to that of

c-myc and hsp70 (Plet, et al. 1995). Some level of pausing is quite common in mammalian genes

composed of a variety of upstream and core promoter elements (Krumm et al. 1995; Blau et al.

1996). This generality of pausing indicates that promoter-associated pausing is an integral feature

of promoters. Perhaps the early phase of elongation is a generally a slow process and in some

genes the property is exploited as a major point of regulation.

Promoter Architecture in Yeast: Is there Pausing?

The power of yeast genetics makes yeast a particularly attractive system for investigating

promoter-associated pausing. But does this form of pausing exist in yeast? Our attempts to

demonstrate pausing in yeast have not yielded a definitive clear example of pausing. We have

examined the HSP82 , SSA4 (hsp70) and GAL1 and GAL10 genes for transcription bubbles

associated with pausing by potassium permanganate assays both before and after induction of the

genes (Giardina and Lis 1993;Giardina and Lis 1995) and (Giardina and Lis, unpubl. results).

Unlike Drosophila heat shock genes, we see no evidence of polymerase on the uninduced genes by

this assay or by the nuclear-run on assay (D. Lee and Lis, unpubl. results). Also, in contrast with

Drosophila hsp70, very little, if any, TBP is associated with the uninduced gene. The reason for

the absence of obvious pausing in the yeast heat shock homologues of Drosophila genes is not

clear. Perhaps yeast does not have to build a potentiated promoter because the chromatin is

generally more accessible to transcription factors. The lack of a bona fide linker histone could lead

to a generally less compact and more accessible chromatin.

After activation of these heat shock and GAL genes, TBP and Pol II are recruited and Pol II

dependent permanganate hypersensitive regions are clearly visible on these promoters. These have

the appearance of the transcription bubble seen with paused Pol II on Drosophila heat shock genes.

This region begins at the same position relative to the TATA box in both Drosophila and yeast. In

the case of Drosophila, this region extends to the pause site; however, in yeast, it extends to the

transcription start site which is further downstream and less precisely positioned in yeast than in

higher eukaryotes (Giardina and Lis 1993;Giardina and Lis 1995). Perhaps Pol II enters and

begins its interaction with the promoters of both higher eukaryotes and yeast by a similar

mechanism, positioned by TBP/TATA box complex. Such a model is in agreement with the

distance (equivalent to 32 bp of B-form DNA) between the yeast Pol II active site and the site of

TFIIB binding derived from two-dimensional crystallography of TFIIB-Pol II complexes of

Kornberg and colleagues (Leuther et al. 1996). Subsequent biochemical events, such as initiation,

are clearly different in yeast relative to higher eukaryotes, and these may account for differences in

the observed melting relative to the sites of initiation. While there is additional permanganate

hypersensitivity after the transcription start site in the activated yeast genes we have examined, this

hypersensitivity is less prominent than that seen upstream of the start site. Other reports to date of

pausing in yeast have been made but are plagued with technical problems. Thus, the issue of

whether pausing exists in yeast remains an open question. While yeast would be an attractive

system to study paused Pol II, the recent increase in sequence information and development of

genetic tools in higher eukaryotes should also allow the rigorous genetic and biochemical

dissection of pausing in vivo in more complex systems as well.

Acknowledgments

I thank the following past and present lab members for making this review possible by their critical

contributions to the analysis of paused polymerase and heat shock promoter architecture and

function (in historical order): Dave Gilmour, Nancy Costlow Lee, Ann Rougvie, Jeff Simon, Bob

Glaser, Janis Werner, Ed Wong, Xiao Hua, Hyun-sook Lee, Tom O'Brien, Eric Rasmussen, Olga

Perisic, Mary Fernandez, Charlie Giardina, Merce Perez-Riba, Lindsay Shopland, Chris Wilkins,

Kazunori Hirayoshi, Adam Law, Paul Mason, Ernie Guzman, Janine Lin, Brian Schwartz, and

Dong-ki Lee.

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Eukaryotic Transcription Provides a Rich Repertoire of Regulatory TargetsFigure 1: Steps in Early TranscriptionA Case for Regulation at the Level of Promoter EscapeFigure 2: Summary of Four Different Classes of Experiments Describing the Paused PolII on the Drosophila Hsp70 GeneTBP and GAGA Factor (GAF) Also Occupy the Uninduced Hsp70 PromoterDNA Sequences Critical in Establishing and Activating the Potentiated PromoterFIgure 3: Model Depicting the Hsp70 Promoter Architecture Before and After Heat Shock ActivationWhy Build a Potentiated Promoter? Speed and AccessMechanism of Transcription Action: PolII Recruitment is Not SufficientPromoter-Associated Pausing is Somewhat General: c-myc, c-fos, and othersPromoter Architecture in Yeast: Is There Pausing?AcknowledgmentsReferences