General Transcription Factors

Chapter 11

False-color transmission electron micrograph of RNAs being synthesized on a DNA template, forming a feather-like structure.

Table of contentsTable of contents

•Class II FactorsClass II Factors•Class I FactorsClass I Factors•UBFUBF•Class III FactorsClass III Factors

11.1 11.1 Class II FactorsClass II Factors

• The class II preinitiation complexThe class II preinitiation complex• Structure and function of TFIIDStructure and function of TFIID• Structure and function of TFIIA and Structure and function of TFIIA and

TFIIBTFIIB• Structure and function of TFIIFStructure and function of TFIIF• Structure and function of TFIIE and Structure and function of TFIIE and

TFIIHTFIIH• Elongation FactorsElongation Factors• The polymerase II holoenzymeThe polymerase II holoenzyme

11.1.1 11.1.1 The class II preinitiation The class II preinitiation complexcomplex

• The general transcription factors The general transcription factors combine with RNA polymerase to combine with RNA polymerase to form a preinitiation complex;form a preinitiation complex;

• Six general transcription factors Six general transcription factors named TFIIA, TFIIB, TFIID, named TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH;TFIIE, TFIIF, and TFIIH;

• The factors and poly II bind in a The factors and poly II bind in a specific order to growing specific order to growing preinitiation complex.preinitiation complex.

Formation of a complex involving Formation of a complex involving TFIID,TFIID,TFIIA,and a promoter-bearing TFIIA,and a promoter-bearing DNADNA Figure 11.1 Formation of a

complex involving TFIID, TFIIA, and a promoter-bearing DNA.

Sharp and coworkers mixed a labeled DNA fragment containing the adenovirus major late promoter with TFIIA and TFIID separately and together, then electrophoresed the products. Lane A, with DNA and TFIIA alone, showed only free DNA, which migrated rapidly, almost to the bottom of the gel. Lane D, with DNA and TFIID alone, showed free DNA plus a non-specific complex (NS). Lane A+D, with both transcription factors, showed a larger complex with both factors (A+D, later named DA).

DNase footprinting the DA DNase footprinting the DA complexcomplex

Figure 11.2 DNase footprinting the DA complex.

Sharp and colleagues performed DNase footprinting with TFIIA, TFIID, and a labeled fragment of DNA containing a TATA box. Lanes 1 and 2 contained sequencing ladders (G+A and G, respectively) obtained by Maxam-Gilbert sequencing of the same DNA fragment. Lane 3 (also denoted F, for "free DNA") was a control with DNA but with no protein added. Lane 4 contained DNA plus TFIID, which presumably formed a non-specific complex (NS). Lane 5 contained DNA plus TFIID and TFIIA (A+D). The footprint in lane 5, indicated with a bracket at right, encompasses the TATA box, which is centered around position -25. The arrow at the top of the bracket denotes a site of enhanced DNase sensitivity adjacent to the protected region.

Building the preinitiation Building the preinitiation complexcomplex

Figure 11.3 Building the preinitiation complex.

Figure 11.3 Building the preinitiation complex. (a) the DABPolF complex. Reinberg and colleagues performed gel mobility shift assays with TFIID, A, B, and F, and RNA polymerase II, along with labeled DNA containing the adenovirus major late promoter Lane 1 shows the familiar DA complex, formed with TFIID and A Lane 2 demonstrates that adding TFIIB caused a new complex, DAB, to form Lane 3 contained TFIID, A, B, and F, but it looks identical to lane 2. Thus, TFIlF did not seem to bind in the absence of polymerase II Lanes 4-7 show what happened when the invesbgators added more and more polymerase II in addibon to the four transcription factors: More and more of the large complexes, DABPolF and DBPolF, appeared. Lanes 8-11 contained less and less TFIIF, and we see less and less of the large complexes. Finally, lane 12 shows that essentially no DABPolF or DBPolF complexes formed when TFIIF was absent, Thus, TFIIF appears to bring polymerase II to the complex. The lanes on the right show what happened when Reinberg and colleagues left out one factor at a time. In lane 13, without TFIID, no complexes formed at all Lane 14 shows that the DA complex, but no tubers, formed in the absence of TFIIB Lane 15 demonstrates that DBPolF could still develop without TFIIA. Finally, all the large complexes appeared in the presence of all the factors (lane 16). (b) The DBPolFEHJA complex Reinberg and colleagues started with the DBPolF complex (lacking TFIIA, lane 1 ) assembled on a labeled DNA containing the adenovirus major late prommer Next, they added TFIIE, then TFIIH, then TFIIJ, then TFIIA, in turn, and performed gel mobility shift assays. With each new transcription factor, the complex grew larger and its mobility decreased further. The mobilities of all the complexes are indicated at right. Lanes 5 -7 show the result of adding more and more TFIIA to the DBPolFEHJ complex, but most of the DBPolFEHJA complex had already formed, even at the lowest TFIIA concentration Lanes 8 -11 show again the resud of leaving out radons factors, denoted at the top of each lane At best, only the DB complex forms At worst, in the absence of TFIID, no complex at all forms.

Footprinting the DA and DAB Footprinting the DA and DAB complexescomplexes Figure 11.4 Footprinting

the DA and DAB complexes.

Reinberg and coworkers performed fooprinting on the DA and DAB complexes with both DNase and another DNA strand breaker: a 1 ,10 phenanthroline-copper ion complex (OP-Cu+). (a) Footprinting on the nontemplate strand. The DA and DAB complexes formed right over the TATA box (TATAAA, indicated at right, top to besom) (b) Footgrinting on the template strand. Again, the protected region in beth the DA and DAB complexes was centered on the TATA box (TATAAA, indicated at right, bottom to top) The arrow near the top at right denotes a site of enhanced DNA cleavage at position +10.

Footprinting the DABPolF Footprinting the DABPolF complexcomplex

Figure 11.5 Footprinting the

DABPolF complex.

Reinberg and colleagues

performed DNase footprinting with

TFIID, A, and B (lane 2) and with

TFIID, A, B, and F, and RNA

polymerase II (lane 3). When RNA

polymerase and TFIIF joined the

complex, they caused a huge

extension of the footprint, to about

position +17. This is consistent with

the large size of RNA polymerase II

Model for formation of the Model for formation of the DABPolF complexDABPolF complex

Figure 11.6 Model for formation of the DABPolF complex. TFIIF (green) binds to polymerase II (Pol II, red) and carries it to the DAB complex. The result is the DABPolF complex. This model conveys the conclusion that polymerase II extends the DAB footprint in the downstream direction, and therefore binds to DNA downstream of the binding site for TFIID, A, and B, which centers on the TATA box.

11.1.2 11.1.2 Structure and Structure and Function Function of TFIIDof TFIID

• TATA Box binding Protein TATA Box binding Protein (TBP)(TBP)

• TBP-associated factors(TAF)TBP-associated factors(TAF)

Methylation interference at Methylation interference at the TATA boxthe TATA box

Figure 11.8 Methylation interference at the TATA box.

Figure 11.8 Methylation interference at the TATA box.

Roeder and colleagues end-labeled DNA containing the adenovirus major late promoter on either the template (a) or nontemplate strand (b), then methylated the DNA under conditions in which As were preferentially methylated. Then they added TFIID and filtered the protein-DNA complexes. DNAs that could still bind TFIID were retained, while free DNA flowed through. Finally, they cleaved the filter-bound and free DNAs at methylated sites with NaOH and subjected the fragments to gel electrophoresis. The autoradiographs in (a) and (b) show that the bound DNA did not cleave in the TATA box, so it was not methylated there. On the other hand, the free DNA was cleaved in the TATA box, showing that it had been methylated there. That is why it no longer bound TFIID. (c) Summary of methylated bases in the free DNA fractions. The lengths of the bars show the intensities of the bands in the "free" lanes in parts (a) and (b), which indicate the degree of methylation. Most of the methylation occurred on As, rather than Gs. These methyl groups are in the minor groove; since this methylated DNA was incapable of binding TFIID, these results suggest that TFItD binds in the minor groove. In reading the sequences in this and the next figure, remember that the nontemplate strand contains the TATA sequence.

Effect of substituting dU for Effect of substituting dU for dT on TFIID binding to the dT on TFIID binding to the TATA boxTATA box

Figure 11.9 Effect of substituting dU for dT on TFIID binding to the TATA box.

Figure 11.9 Effect of substituting dU for dT on TFIID binding

to the TATA box.

Roeder and coworkers bound TWIID to labeled DNA containing

TATA boxes with the sequences given at top. They did the binding in

the presence of excess unlabeled competitor DNA containing either

wild-type or mutant TATA boxes (mutant sequence: TAGAGAA). To

assay for TFIID-TATA box binding, they electrophoresed the protein-

DNA complexes under non-denaturing conditions which separate

free DNA from protein-bound DNA. In all cases, the wild-type TATA

box was able to compete, so only free DNA was observed (even-

numbered lanes). However, in all cases, the mutant TATA box was

unable to compete, even when the labeled TATA box contained a dU

instead of a dT. In fact, lane 7 shows that substitution of a dU for a

dT in position 2 of the template strand of the TATA box (sequence:

AdUATTTT) actually seemed to enhance TFIID-TATA box binding

compared to the unsubstitued TATA box (lane 1 ). Since dU and dT

differ in the major groove, but not the minor groove, and the

substitution of dU for dT did not inhibit binding, this suggests that

TFIID binds in the minor groove.

Effect of substituting C for T Effect of substituting C for T and I for A on TFIID binding to and I for A on TFIID binding to the TATA boxthe TATA box

Figure 11.10 Effect of substituting C for T and I for A on TFIlD binding to the TATA box.

Figure 11.10 Effect of substituting C for T and I for A on

TFIlD binding to the TATA box. (a) Appearance of nueleosides as viewed from the major and

minor grooves. Notice that thymine and cytidine look identical from the

minor groove (green, below), but quite different from the major groove

(red, above) Similarly. adenosine and inosine look the same from the

minor groove, but very different from the major groove.

(b) Sequence of the adenovirus major late promoter (MLP)

TATA box with Cs substituted for Ts and Is substituted for AS,

yielding a CICI box .

(c) Binding TBP to the CICI box. Start and Hawley performed gel

mobility shift assays using DNA fragments containing the MLP with a

CICI box (lanes 1-3) or the normal TATA box (lanes 4-6), or a non-

specific DNA (NS) with no promoter elements (lanes 7-9) The first lane

in each set (1,4, and 7) contained yeast TBP; the second lane in each set

(2, 5, and 8) contained human TSP; and the third lane in each set

contained just buffer The yeast and human TBPs gave rise to slightly

different size brotein-DNA comple~es, but substituting a CICI box for

the TATA box had little effect on the yield of the complexes. Thus, TBP

binding to the TATA box was not significantly diminished by the

substitutions.

Structure of the TBP-TATA Structure of the TBP-TATA box complexbox complex

Figure 11.6 Structure of the TBP-TATA box complex.

This diagram, based on Sigler and colleagues' crystal structure

of the TBP-TATA box complex, shows the backbone of the TBP in

olive at top. The long axis of the "saddle" is in the plane of the

page. The DNA below the protein is in multiple colors. The

backbones in the region that interacts with the protein are in

orange, with the base pairs in red. Notice how the protein has

opened up the narrow groove and almost straightened the helical

twist in that region. One stirrup of the TBP is seen as an olive loop

at right center, inserting into the minor groove. The other stirrup

performs the same function, but it is out of view in back of the

DNA. The two ends of the DNA, which do not interact with the

TBP, are in blue and gray: blue for the backbones, and gray for the

base pairs. The left end of the DNA sticks about 25 degrees out of

the plane of the page, and the right end points inward by the same

angle. The overall bend of about 80 degrees in the DNA, caused by

TBP, is also apparent.

Figure 11.7 Effects of mutations in TBP on transcription by all three RNA polymerases.

Figure 11.7 Effects of mutations in TBP on transcription by all

three RNA polymerases.

(a) Locations of the mutations. The boxed region indicates the conserved

C-terminal domain of the TBP; red areas denote two repeated

elements involved in DNA binding. The two mutations are: P65 →S, in

which proline 65 is changed to a serine; and 1143 → N, in which

isoleucine 143 is changed to asparagine. (b-e) Effects of the

mutations. Reeder and Hahn made extracts from wild-type or mutant

yeasts, as indicated at bottom, and either heat-shocked them at 37

℃or left them at 24℃, again as indicated at bottom. Then they tested

these extracts by S1 analysis for ability to start transcription at

promoters recognized by all three nuclear RNA polymerases:

(b) The rRNA promoter (polymerase I); (c) the CYC1 (polymerase II)

promoter; (d) the 5S rRNA promoter (polymerase III); and (e) the

tRNA promoter (also polymerase III). The 1143 →N extract was

deficient in transcribing from all four promoters even when not heat-

shocked. The P65 →S extract was deficient in transcribing from

polymerase II and III promoters, but could recognize the polymerase

promoter, even after heat shock.

SUMMARYSUMMARY

TFIID contains a 38 kDa TATA box-binding

protein (TBP) plus several other polypeptides

known as TBP-associated factors (TAFIIs). The C-

terminal 180 amino acid fragment of the human

TBP is the TATA box-binding domain. The

interaction between a TBP and a TATA box is an

unusual one that takes place in the DNA minor

groove. The saddle-shaped TBP lines up with the

DNA, and the under-side of the saddle forces

open the minor groove and bends the TATA box

into an 80°curve.

Structure of a Structure of a DrosophilaDrosophila TFIID assembled inTFIID assembled in vitro vitro from from the products of cloned genesthe products of cloned genes

Relationships among the Relationships among the TAFs of fruit TAFs of fruit flies,humans,and yeastflies,humans,and yeast

Figure 11.13 relationships among

the TAFs of fruit flies

(D.melanogaster), humans (H.

sapiens), and yeast (S. cerevisiae).

The horizontal lines link homologous

proteins.

Activities of TBF and TFIID on Activities of TBF and TFIID on four different promotersfour different promoters

Figure 11.14 Activities of TBP and TFIID on four different promoters.

Tjian and colleagues tested a reconstituted Drosophila transcription system containing either TBP or TFIID (indicated at top) or templates bearing four different promoters (also as indicated at top). The promoters were of two types diagrammed at bottom: The first type, represented by the adenovirus E1B and E4 promoters, contained a TATA box (red). The second type, represented by the adenovirus major late promoter (AdML) and the Drosophila Hsp70 promoter, contained a TATA box plus an initiator (I, green) and a downstream element (D, blue). After transcription in vitro, Tjian and coworkers assayed the RNA products by primer extension (top). The autoradiographs show that TBF and TFIID fostered transcription equally well from the first type of promoter (TATA box only), but that TFIID worked much better than TBP in supporting transcription from the second type of promoter (TATA box plus initiator plus downstream element).

Identifying the TAFIdentifying the TAFIIIIs that s that bind to the bind to the hsp70hsp70 promoter promoter

Figure 11.15 Identifying the TAFIIs that bind to the hsp70 promoter. Tjian and colleagues photo-crosslinked TFIID to a 32p-labeled template containing the hsp70 promoter. This template had also been substituted with the photo-sensitive nucleoside bromodeoxyuridine (BrdU). Next, these workers irradiated the TFIID-DNA complex with ultraviolet (UV) light to form covalent bonds between the DNA and any proteins in close contact with the major groove of the DNA. Next, they digested the DNA with nuclease and subjected the proteins to SDS-PAGE. Lane 1 of the autoradiograph shows the results when TFIID was the input protein. TAFII250 and TAFII150 became labeled, implying that these two proteins had been in close contact with the labeled DNA's major groove. Lane 2 is a control with no TFIID. Lane 3 shows the results when a ternary complex containing TBP, TAFII250, and TAFII150 was the input protein. Again, the two TAFIIs became labeled, suggesting that they bound to the DNA. Lane 4 shows the results when TBP was the input protein. It did not become labeled, which was expected since it does not bind in the DNA major groove.

DNase I footprinting the DNase I footprinting the hsp70hsp70 promoter with TBP and the promoter with TBP and the ternary complexternary complex

Figure 11.16 DNase I footprinting the

hsp70 promoter with TBP and the

ternary complex (TBP, TAFII250, and

TAFII150).

Lane 1, no protein; lane 2, TBP; lane 3,

ternary complex. In both lanes 2 and 3, TFIIA

was also added to stabilize the DNA-protein

complexes, but separate experiments

indicated that it did not affect the extent of

the footprints. Lane 4 is a Maxam-Gilbert

G+A sequencing lane used as a marker. The

extents of the footprints caused by TBP and

the ternary complex are indicated by

brackets at left. The locations of the TATA

box and initiator are indicated by boxes at

right.

Model for the interaction Model for the interaction between TBP and TATA-between TBP and TATA-containing or TATA-less containing or TATA-less promoterspromoters

Failure of TBP alone to Failure of TBP alone to respond to Sp1respond to Sp1

Figure 11.18 Failure of TBP alone to respond to Sp1.

(a) Structure of the test promoter. This is a composite Sp1-responsive promoter containing six GC boxes (red) from the SV40 early promoter and the TATA box (blue) and transcription start site (initiator, green) from the adenovirus major late promoter. Accurate initiation from this promoter in the run-off assay described below should produce a 375 nt transcript. (b) In vitro transcription assay. Tjian and colleagues mixed TFIID, or bhTBP, or vhTBP, as shown at top, with TFIIA, TFIIB, TFIIE, TFIIF, and RNA polymerase II, then performed a run-off transcription assay with [α- 32p] UTP. Lanes 1 and 2 show that natural TFIID supported a high level of transcription from this promoter, and this transcription was significantly enhanced by the transcription factor Sp1. Lanes 3-6 demonstrate that any transcription due to recombinant human TBP was not stimulated by Sp1 in the absence of TAFIIs.

Activation by Sp1 requires Activation by Sp1 requires TAFTAFIIII110110

Figure 11.19 Activation by Sp1 requires TAFII110.

Tjian and colleagues used a primer extension assay to measure transcription from a template containing a TATA box and three upstream GC boxes. They used either a Drosophlia cell extract (a) or a human cell extract (b), each of which had been depleted of TFIID. They replaced the missing TFIID with any of the three different complexes, picture at bottom, containing combinations of TBP, TAFII250, and TAFII110. They also added no Sp1 (-), or two increasing concentrations of Sp1, represented by the wedges. The autoradiographs show the amount of transcription, and therefore the activation achieved by Sp1 with each set of TAFIIs. Activation was observed in each extract only with all three TAFIIs.

A model for transcription enhancement A model for transcription enhancement

by activatorsby activators Figure 11.20 A model for

transcription enhancement by activators.

(a) TAFII250 does not interact with

either Sp1 or Gal4-NTF-1 (a hybrid activator with the transcription-activating domain of NTF-1), so no activation takes place.

(b) Gal4-NTF-1 can interact with either

TAFII150 or TAFII60 and activate

transcription; Sp1 cannot interact with either of these TAFs or with TAFII250 and does not activate transcription.

(c) Gal4-NTF-1 interacts with TAFII150

and Sp1 interacts with TAFII110,so

both factors activate transcription.

(d) Holo TFIID contains the complete

assortment of TAFIIs, so it can respond

to a wide variety of activators, represented here by Sp1, Gal4-NTF-1, and a generic activator at top.

Whole genome analysis of Whole genome analysis of transcription requirements transcription requirements

in yeastin yeastGeneral transcription Fraction General transcription Fraction of genes of genes

Factor (subunit) Factor (subunit) Dependent on Dependent on

Subunit Subunit function(%)function(%)

TFIID (TAFTFIID (TAFIIII145) 16145) 16

TFIID (TFTFIID (TFIIII17) 6717) 67

TFIIE (Tfa1) 54TFIIE (Tfa1) 54

TFIIH (Kin28) 87TFIIH (Kin28) 87

Figure 11.18 Three-dimensional models of TFIID and TFTC. Schultz and colleagues made negatively stained electron micrographs (see Chapter 19, for method) of TFIID and TFTC, then digitally combined images to arrive at an average. Then they tilted the grid in the microscope and analyzed the resulting micrographs to gleanthree-dimensional information for both proteins. The resulting models for TFIID (green) and TFTC (blue) are shown.

SUMMARYSUMMARY TFIID contains at least eight TAFIIs, in addition to

TBP. Most of these TAFIIs are evolutionarily

conserved in the eukaryotes. The TAFIIs serves

several functions, but two obvious ones are interacting with core promoter elements and interacting with gene-specific transcription factors. TAFII250 and TAFII150 help TFIID bind to the initiator

and downstream elements of promoters and therefore can enable TBP to bind to TATA-less promoter that contain such elements. TAFII250 and TAFII110 help

THIID interact with Sp1 that is bound to GC boxes upstream of the transcription start site. These TAFIIs

therefore ensure that TBP can bind to TATA-less promoters that have GC boxes. Different combinations of TAFIIs are apparently required to

respond to various transcription activators, at least in higher eukaryotes. TAFII250 also has two enzymatic

activities. It is a histone acetyl trans

11.1.3 11.1.3 Structure and Structure and function of TFIIA and function of TFIIA and

TFIIBTFIIB• TFIIATFIIA: 2-3 subunits, : 2-3 subunits,

binds to TBP and stabilizes binds to TBP and stabilizes

binding between TFIID and binding between TFIID and promoters;promoters;

• TFIIBTFIIB: a linker between TFIID and : a linker between TFIID and

TFIIF/polymeraseTFIIF/polymerase

Hypothetical structure of a TFIIA-TFIIB-TBP-TATA box complexHypothetical structure of a TFIIA-TFIIB-TBP-TATA box complex

Figure 11.19 Hypothetical structure of a TFIIA-TFIIB-TBP-TATA box complex.

This is a combination of two structures: a human core TFIIB-plant TBP-adenovirus TATA box structure, and a yeast TFIIA-TBP-TATA box structure. None of the proteins is complete. They are all core regions that have the key elements needed to do their jobs. The DNA is gray; the two halves of core TBP are light blue (upstream half) and dark blue (downstream half); the amino terminal domain of core TFIIB is red and the carboxyl terminal domain is magenta; the core large subunit of TFIIA is green, and the small subunit is yellow. The transcription start site is at right, denoted "+1 ."

SUMMARYSUMMARY THIIA contains two subunits (yeast), or three subunits (fruit flies and humans). This factor is probably more properly considered a TAFII since it binds to TBP and stabilizes binding between TFIID and promoters. TFIIB serves as a linker between TFIID and TFFIIF/ polymerase II. It has two domains, one of which is responsible for binding to TFIID, the other for continuing the assembly of the preinitiation complex. A structure for the TFIIA-TFIIB-TBP-TATA box complex can be imagined, based on the known structures of the TFIIA-TBP-TATA box and TFIIB-TBP-TATA box complexes. This structure shows TFIIA and TFIIB binding to the upstream and downstream stirrups, respectively, of TBP. This puts these two factors in advantageous positions to perform their functions.

11.1.4 11.1.4 Structure and function Structure and function of TFIIFof TFIIF

Binding of the polymerase to the DAB complex requires prior interaction with TFIIF, composed of two polypeptides called RAP30 and RAP70. RAP30 is the protein that ushers polymerase into the growing complex.

Role of TFIIF in binding RNA Role of TFIIF in binding RNA polymerase II to the polymerase II to the preinitiation complexpreinitiation complex

Figure 11.22 Role of TFIIF in binding RNA polymerase II to the preinitiation complex.

Figure 11.22 Role of TFIIF in binding RNA polymerase II to the preinitiation complex.

Greenblatt, Reinberg, and colleagues performed phenyl-

Superose micro column chromatography on TFIIF and tested fractions

for (a) TFIIF transcription factor activity;

(b) preinitiation complex formation with RNA polymerase II, using a

gel mobility shift assay; and (c) content of RAP30, detected by

Western blotting and probing with an anti-RAP30 antibody. (a) TFIIF

activity assay. Lane I, activity of the protein loaded onto the column

(input); lane +, positive control with known TFIIF activity; other lanes

are numbered according to their order of elution from the column.

The great majority of the TFIIF activity eluted in fractions 16-22. (b)

Gel mobility shift assay. The lanes on the left show the complexes

formed with the TFIIF input fraction alone (I), and with various

combinations of

highly purified TFIID, A, B, polymerase II, and TFIIF. The numbered

lanes show the shifts in the DAB complex produced by addition of

polymerase II plus the same column fractions as in part (a). The ability

to form the DABPolF complex resided in the same fractions with TFIIF

activity (16-22). (c) Western blot to detect RAP30. The labeling of the

lanes has the same meaning as in panel (a). The fractions with RAP30

(16-22) were the same ones with TFIIF activity and the ability to bring

polymerase II into the preinitiation complex. Thus, RAP30 seems to

have this activity.

11.1.5 11.1.5 Structure and Structure and function of TFIIE and function of TFIIE and

TFIIHTFIIH

Formation of the DABPoIFE Formation of the DABPoIFE complexcomplex

Figure 11.23 Formation of the DABPolFE complex.

Figure 11.23 Formation of the DABPolFE complex.

Tjian, Reinberg, and colleagues performed gel mobility shift assays with various combinations of transcription factors, polymerase II, and a DNA fragment containing the adenovirus major late promoter. The protein components in each lane are given at top, and the complexes formed are indicated at left. Note that TFIID, A, B, F, E, and polymerase II formed the DABPolFE complex, as expected (lane 4). Lanes 5-8 show that increasing quantities of the two subunits of TFIIE, added separately, cannot join the DABPolF complex. However, lanes 9 and 10 demonstrate that the two polypeptides can join the complex if they are added together. Lane 11 is a repeat of lane 10, and lane 12 is identical except that it is missing TFIID. This is a reminder that everything depends on TFIID, even with all the other factors present.

Dependence of Dependence of transcription on both transcription on both subunits of TFIIEsubunits of TFIIE

Figure 11.24 Dependence of transcription on both subunits of TFIIE.

Figure 11.24 Dependence of transcription on both subunits

of TFIIE.

(a) Tjian and Reinberg performed run-off transcription of a DNA

fragment containing the adenovirus major late promoter in the

presence of all transcription factors except TFIIE. They added

whole TFIIE or the products of cloned genes encoding the subunits

of the transcription factor in increasing concentration, as indicated

at top. The wedge shapes illustrate the increase in concentration of

each factor from one lane to another. Lanes 1 and 2 show that

native TFIIE can reconstitute transcription activity. However, the

subunits added separately cannot do this, as portrayed in lanes 3-

10. On the other hand, the two subunits together can stimulate

transcription.

(b) The same kind of run-off assays, using the TATA-less G61

promoter, showed that the TFIIE produced by cloned genes

stimulates Sp1-dependent transcription. Lanes 1 and 2 contained

native TFIIE purified from HeLa cells. Lanes 3 and 4 contained

TFIIE subunits produced by cloned genes. Lanes 5 and 6 had no

TFIIE. Clearly, TFIIE is necessary, and the factor made by cloned

genes works as well as the native one. Also, as we have seen before,

transcription of the TATA-less promoter requires Sp1.

The preinitiation complex The preinitiation complex envisioned by Tjian and envisioned by Tjian and ReinbergReinberg

Figure 11.25 The preinitiation complex envisioned by Tjian and Reinberg.

This construct contains air of the factors in the DABPolFE complex plus TFIIH (orange), another general transcription factor we shall discuss next.

Phosphorylation of Phosphorylation of preinitiation complexespreinitiation complexes

Figure 11.26 Phosphorylation of

preinitiation complexes. Reinberg and colleagues performed gel mobility shift assays with preinitiation complexes DAB through DABPolFEH, in the presence and absence of ATP, as indicated at top Only when TFIIH was present did ATP shift the mobility of the complex (compare lanes 7 and 8). The simplest explanation is that TFIIH promotes phosphorylation of the input polymerase (polymerase IIA) to polymerase IIO.

TFIIH phosphorylates RNA TFIIH phosphorylates RNA polymerase IIpolymerase II

Figure 11.21 TFIIH phosphorylates RNA polymerase II.

Figure 11.21 TFIIH phosphorylates RNA polymerase II. (a) Reinberg and colleagues incubated polymerase IIA with various mixtures of transcription factors, as shown at top. They included [γ-32P]ATP in all reactions to allow phosphorylation of the polymerase, then electrophoresed the proteins and performed autoradiography to visualize the phosphorylated polymerase. Lane 4 shows that TFIID, B, F, and E, were insufficient to cause phosphorylation. Lanes 5-10 demonstrate that TFIIH alone is sufficient to cause some polymerase phosphorylation, but that the other factors enhance the phosphorylation. TFIIE provides particularly strong stimulation of phosphorylation of the polymerase IIa subunit to IIo. (b) Time course of polymerase phosphorylation. Reinberg and colleagues performed the same assay for polymerase phosphorylation with TFIID, B, F, and H in the presence or absence of TFIIE, as indicated at top. They carried out the reactions for 60 or 90 min, sampling at various intermediate times, as shown at top. The small bracket at left indicates the position of the polymerase IIo subunit, and the larger bracket shows the locations of IIa and IIo together (IIa/IIo). Arrows also mark the positions of the two polymerase subunit forms. Note that polymerase phosphorylation is more rapid in the presence of TFIIE. (c) Graphic presentation of the data from panel (b). Green and red curves represent phosphorylation in the presence and absence, respectively, of TFIIE. Solid lines and dotted lines correspond to appearance of phosphorylated polymerase subunits IIa and IIo, or just IIo, respectively.

TFIIH phosphorylates the CTD of TFIIH phosphorylates the CTD of polymerase IIpolymerase II

Figure 11.28 TFIIH phosphorylates the CTD of polymerase II. (a) Reinberg phosphorylated increasing amounts of polymerases IIA, IIB, or IIO, as indicated at top, with TFIID, B, F, E, and H and radioactive ATP as described in Figure 11.27. Polymerase liB, lacking the CTD, could not be phosphorylated. The unphosphorylated polymerase IIA was a much better phosphorylation substrate than IIO, as expected. (b) Purification of the phosphorylated CTD. Reinberg and colleagues cleaved the CTD from the phosphorylated polymerase Ila subunit with the protease chymotrypsin (Chym), electrophoresed the products, and visualized them by autoradiography. Lane 1, reaction products before chymotrypsin cleavage; lanes 2 and 3, reaction products after chymotrypsin cleavage. The position of the CTD had been identified in a separate experiment.

Helicase activity of TFIIHHelicase activity of TFIIH

Figure 11.29 Helicase activity of TFIIH. (a) The helicase assay. The substrate consisted of a labeled 41-nt piece of DNA (red) hybridized to its complementary region in a much larger, unlabeled, single-stranded M13 phage DNA (blue). DNA helicase unwinds this short helix and releases the labeled 41-nt DNA from its larger partner. The short DNA is easily distinguished from the hybrid by electrophoresis. (b) Results of the helicase assay. Lane 1, heat-denatured substrate; lane 2, no protein; lane 3, 20 ng of RAD25 with no ATP; lane 4, 10 ng of RAD25 plus ATP; lane 5, 20 ng of RAD25 plus ATP.

The TFIIH DNA helicase gene The TFIIH DNA helicase gene product(RAD25) is required for product(RAD25) is required for transcription in yeasttranscription in yeast

Figure 11.30 The TFIIH DNA helicase gene product (RAD25) is required for transcription in yeast.

Prakash and colleagues tested extracts from wild-type (RAD25) and temperature-sensitive mutant (rad25-ts24) cells for transcription of a G-less cassette template at the permissive (a) and nonpermissive (b) temperatures. After allowing transcription for 0-10 minutes in the presence of ATP, CTP, and UTP (but no GTP), with one 32P-labeled nucleotide, they electrophoresed the labeled products and detected the bands by autoradiography. The origin of the extract (RAD25 or rad25-ts24), as well as the time of incubation in minutes, is given at top. Arrows at left denote the positions of the two G-less transcripts. We can see that transcription is temperature-sensitive when the TFIIH DNA helicase (RAD25) is temperature-sensitive.

A model for the participation A model for the participation of general transcription of general transcription factors in initiationfactors in initiation

Figure 11.31 A model for the participation of general transcription factors in initiation, promoter clearance, and elongation.

(a) TBP (or TFIID), along with TFIIB, TFIIF, and RNA polymerase II form a minimal initiation complex that makes abortive transcripts (magenta) at the initiator, which is melted. (b) TFIIE and TFIIH join the complex, converting it to an active transcription complex. (c) The DNA helicase activity of TFIIH uses ATP to unwind more of the DNA double helix at the initiator. Somehow, this allows promoter clearance. (d) With addition of NTPs, the elongation complex continues elongating the RNA. TBP and TFIIB remain at the promoter: TFIIE and TFIIH are not needed for elongation and dissociate from the elongation complex.

SUMMARYSUMMARY TFIIE, composed of two molecules each of a 34 kDa

and a 56 kDa polypeptide, binds after polymerase and

TFIIF. Both subunits are required for binding and

transcription stimulation. A protein known as MO15/CDK7,

which associates closely with TFIIH, phosphorylates the

carboxyl terminal domain (CTD) of the largest RNA

polymerase 11 subunit. TFIIE greatly stimulates this

process in vitro. TFIIE and TFIIH are not essential for

formation of an open promoter complex, or for elongation,

but they are required for promoter clearance. TFIIH has a

DNA helicase activity that is essential for transcription, at

least in yeast, presumably because it facilitates promoter

clearance.

11.1.6 11.1.6 Elongation factorsElongation factors

Transcription can be controlled

at the elongation level. One factor,

TFIIS, stimulates elongation by

limiting long pauses at discreet

sites TFIIF also stimulates

elongation, apparently by limiting

transient pausing.

Effect of TFIIS on Effect of TFIIS on transcription initiation and transcription initiation and elongation combinedelongation combined

Figure 11.33 Effect of TFIIS on

transcription initiation and

elongation combined.

Reinberg and Roeder carried

out this experiment in the same

manner as in Figure 11.32, except

for the orcer of additions to the

reaction. Here, they added TFIIS (or

buffer) at the beginning instead of

last (see time line at bottom). Thus,

TFIIS had the opportunity

to .stimulate both initiation and

elongation. The dashed vertical lines

show no more stimulation than in

Figure 11.32.

• Transcription can be Transcription can be controlled at the elongation controlled at the elongation level, TFIIS, stimulates level, TFIIS, stimulates elongation by limiting long elongation by limiting long pauses at discrete sites. pauses at discrete sites. TFIIF also stimulates TFIIF also stimulates elongation, apparently by elongation, apparently by limiting transient pausing.limiting transient pausing.

Figure 11.29 A model for proofreading

by RNA polymerase II.

(a) The polymerase, transcribing the DNA

from left to right, has just incorporated an

incorrect nucleotide (yellow).

(b) The polymerase backtracks to the left,

extruding the 3'-end of the RNA, with its

misincorporated nucleotide, out of the

active site of the enzyme. At this point, the

polymerase is irreversibly arrested unless

the extruded RNA can be removed.

(c) The ribonuclease activity of the

polymerase clips off the 3'-end of the RNA,

including the incorrect nucleotide.

(d) The polymerase resumes transcription.

• TFIIS stimulates proofreading—TFIIS stimulates proofreading—the correction of mis-the correction of mis-incorporated nucleotide—incorporated nucleotide—presumably by stimulating the presumably by stimulating the RNase activity of the RNA RNase activity of the RNA polymerase, allowing it to cleave polymerase, allowing it to cleave off a mis-incorporated nucleotide off a mis-incorporated nucleotide (with a few other nucleotides) and (with a few other nucleotides) and replace it with the correct one.replace it with the correct one.

11.1.7 11.1.7 The polymerase II The polymerase II holoenzymeholoenzyme

Yeast and mammalian cells have an RNA

polymerase II holoenzyme that contains

many polypeptide in addition to the subunits

of the polymerase. The yeast holoenzyme

contains a subset of general transcription

factors and at least some of the SRB

proteins. The rat holoenzyme contains all the

general transcription factors and at least

some of the SRB proteins. The rat

holoenzyme contains all the general

transcription factors except TFIIA.

Purified yeast RNA Purified yeast RNA polymerase II holoenzymepolymerase II holoenzyme

Figure 11.34 Purified yeast RNA polymerase II holoenzyme.

Kornberg and colleagues used a purification scheme that included immunoprecipitation to isolate a polymerase II holoenzyme from yeast cells, then subjected the polypeptide constituents of this holoenzyme to SDS-PAGE Lane 2 displays these polypeptides (h -pol II), while lane 1 contains the subunits of the "core RNA polymerase II" (c- pol II) for comparison.

11.2 11.2 Class I FactorsClass I Factors

The preinitiation complex The preinitiation complex that forms at rRNA that forms at rRNA promoters.promoters.

• SL1SL1• Upstream binding factor Upstream binding factor

(UBF)(UBF)

11.2.1 11.2.1 SL1SL1

SL1 plays a role in SL1 plays a role in assembling the assembling the polymerase I polymerase I preinitiation factor.preinitiation factor.

SL1 is a species-specific SL1 is a species-specific transcription factortranscription factor

Figure 11.35 SL1 is a species-specific transcription factor.

Tjian and colleagues performed a run-off assay with a mouse cell-free extract and two templates, one containing a mouse rRNA promoter, the other containing a human rRNA promoter. The mouse and human templates gave rise to run-off transcripts of 2400 and 1500 nt, respectively. As shown at bottom, lane 1 contained no human SL1, and essentially only the mouse template was transcribed. As Tjian and colleagues added more and more human SL1, they observed more and more transcription of the human template, and less transcription of the mouse template. In lane 5, transcription of both templates seems to be suppressed.

Footprinting the rRNA Footprinting the rRNA promoter with SL1 and RNA promoter with SL1 and RNA polymerase polymerase

Figure 11.36 Footprinting the rRNA promoter with SL1 and RNA polymerase I.

Figure 11.36 Footprinting the rRNA promoter with SL1 and RNA polymerase I.

Tjian and colleagues performed DNase I footprinting with either the nontemplate strand (a), or the template strand (b) of the human rRNA promoter They added SL1 and/or RNA polymerase, as indicated at bottom. Brackets indicate footprint regions, while stars designate sites of enhanced DNase sensitivity Polymerase I by itself can protect a region (A) of the UCE; polymerase and SL1 together extend the protection into another region (B) of the UCE. Binding of SL1 by itself is not detectable by this assay (c) Summary of footprints. Bars above and below the UCE region represent the footprints on the template and nontemplate strands. respectively, with the A and B sections delineated. Again, stars represent the sites of enhanced cleavage.

The core promoter element determines The core promoter element determines species specificityspecies specificity

Figure 11.37 The core promoter element determines species specificity.

Figure 11.37 The core promoter element determines species specificity.

Tjian and colleagues constructed human, mouse,and hybrid human/mouse rRNA premofers and tested them for promoter activily by a run-off transcription assay with partially purified human RNA polymerase I and highly purified human SL1. All reactions contained a control template, △5'/-83, which had a human rRNA promoter lacking the UCE. This gave a basal level of transcription in all cases and could be used to normalize the reactions. The expected position of each run-off transcript is indicated at left with an arrow. The first two lanes in each set of three contained increasing quantities of human SL1. as indicated by the "wedges"; the third lane in each set had no SL1. Diagrams of each construct are given at right. Human promoter elements are rendered in green, and mouse elements in pink Only when the construct contained a human core element did transcription occur. The nature of the UCE was irrelevant. Human SL1 was also required. Thus, the core element determines the species-specificity of the rRNA promoter.

11.2.2 11.2.2 UBFUBF

• Stimulates transcription by Stimulates transcription by polymerase I;polymerase I;

• Actives the intact promoter Actives the intact promoter or the core element;or the core element;

• Mediates activation by UCEMediates activation by UCE

Interaction of UBF and SL1 Interaction of UBF and SL1 with the rRNA promoterwith the rRNA promoter

Figure 11.38 Interaction of UBF and SL1 with the rRNA promoter.

Tjian and colleagues performed DNase I footprinting with the human rRNA promoter and various combinations of polymerase I + UBF and SL1 (a), or UBF and SL1 (b) The proteins used in each lane are indicated at bottom. The positions of the UCE and core elements are shown at left, and the locations of the A and B sites are illustrated with brackets at right Stars mark the positions of enhanced DNase sensitivity SL1 caused no footprint on its own, but enhanced and extended the footprints of UBF in both the UCE and the core element This enhancement is especially evident in the absence of polymerase I (panel b).

Activation of transcription Activation of transcription from the rRNA promoter by from the rRNA promoter by SL1 and UBFSL1 and UBF

Figure 11.39 Activation of transcription

from the rRNA promoter by SL1 and

UBF.

Tjian and colleagues used an S1 assay to

measure transcription from the human

rRNA promoter in the presence of RNA

polymerase I and various combinations of

UBF and SL1, as indicated at top, The top

panel shows transcription from the wild-type

promoter; the bottom panel shows

transcription from a mutant promoter (△5' -

57) lacking UCE function. SL1 was required

for at least basal activity, but UBF enhanced

this activity on both templates.

Effect of mutations in the UCE on UBF Effect of mutations in the UCE on UBF

activationactivation

(a)Description of mutants and effects on binding(a)Description of mutants and effects on binding

Inserted linkers are represented by boxes, deletions by spaces, and Inserted linkers are represented by boxes, deletions by spaces, and

bases altered following site-directed mutagenesis by Xs. The positions of bases altered following site-directed mutagenesis by Xs. The positions of

sites A and B of the UCE, relative to the mutations, are given at bottom. sites A and B of the UCE, relative to the mutations, are given at bottom.

Binding of UBF, or UBF/SL1. to each mutant promoter is reported at right. Binding of UBF, or UBF/SL1. to each mutant promoter is reported at right.

Tjian and colleagues measured the binding by footprinting; the criterion for Tjian and colleagues measured the binding by footprinting; the criterion for

UBF/Shl binding was extension of the footprint into site B. UBF/Shl binding was extension of the footprint into site B.

(b)Effect on transcription(b)Effect on transcription

(b) Effect on transcription. Tjian and Coworkers measured transcription by 81 analysis as in Figure 11.39, in the presence (right panel) or absence (left panel) of UBF. They included SL1 and polymerase I in all cases. They also added a pseudo wild-type template (ψWT) as an internal control in all cases. The nature of the test template (wild-type or mutant) is given at the top of each lane. Mutant-186/-163 behaved like the wild-type template in that it supported stimulation by UBF. By contrast, all the other mutant templates were considerably impaired in ability to respond to UBF.

11.2.3 11.2.3 The Universality of The Universality of TBPTBP

Effect of mutations in TBP on Effect of mutations in TBP on transcription by all three RNA transcription by all three RNA

polymerasespolymerases

11.2.4 11.2.4 Structure and Structure and function of SL1function of SL1

SL1 is composed of TBP and

three TAFs, TAFI110, TAFI63, and

TAFI48. Fully functional and species-

specific SL1 can be reconstituted

from these purified components, and

binding of TBP to the TAFIs precludes

binding to the TAFIIS.

Co-purification of SL1 Co-purification of SL1 and TBPand TBP

Figure11.42 Co-purification of SLl and TBP.

Figure11.42 Co-purification of SLl and TBP.

(a) Heparin agarose column chromatography Top:

Pattern of elution from the column of total protein (red)

and salt concentration (blue), as well as three specific

proteins (brackets). Middle: SL1 activity, measured by

S1 analysis, in selected tractions Bottom: TBP protein,

detected by Western blotting, in selected fractions Both

SL1 and TBP were centered around fraction 56

(b) Glycerol gradient ultracentrifugation Top:

Sedimentation profile of TBP Two other proteins,

catalase and aldolase, with sedimentation coefficients

of 11.3 S and 73 S, respectively, were run in a parallel

centrifuge tube as markers Middle and bottom panels,

as in pad (a) Both SL1 and TBP sedimented to a

position centered around fraction 16.

Immunodepletion of TBP Immunodepletion of TBP inhibits SL1 activityinhibits SL1 activity

The TAFs in SL1The TAFs in SL1

Figure 11.44 The TAFs in SL1.

Tjian and colleagues immunoprecipitated SL1 with an anti-TBP antibody and subjected the polypeptides in the immunoprecipitate to SDS-PAGE. Lane 1, molecular weight markers; lane 2, immunoprecipitate (IP); lane 3, purified TBP for comparison; lane 4, another sample of immunoprecipitate; lane 5, TFIID TAFs (PolII-TAFs) for comparison; lane 6, pellet after treating immunoprecipitate with 1 M guanidine-HCl and re-precipitating, showing TBP and antibody; lane 7, supernatant after treating immunoprecipitate with 1 M guanidine-HCl and reprecipitating, showing the three TAFs (labeled at right).

11.3 11.3 Class III FactorsClass III Factors

• TFIIIATFIIIA• TFIIIB and CTFIIIB and C• The role of TBPThe role of TBP

Transcription of all class III Transcription of all class III genes requires TFIIIB and C, genes requires TFIIIB and C, and Transcription of the 5S and Transcription of the 5S rRNA genes requires these two rRNA genes requires these two plus TFIIIAplus TFIIIA..

11.3.1 TFIIIA11.3.1 TFIIIA

Effect of anti-TFIIIA on Effect of anti-TFIIIA on transcription by polymerase transcription by polymerase

IIIIIIFigure 11.45 Effect of anti-TFIIIA on

transcription by polymerase III.

Brown and colleagues added cloned 5S

rRNA and tRNA genes to an oocyte extract (a), or

a somatic cell extract (b) in the presence of labeled

nucleotide and: no antibody (lanes 1), an irrelevant

antibody (lanes 2), or an anti-TFIIIA antibody

(lanes 3). After transcription, these workers

electrophoresed the labeled RNAs. The anti-TFIIIA

antibody blocked 5S rRNA gene transcription in

both extracts, but did not inhibit tRNA gene

transcription in either extract. The oocyte extract

could process the pre-tRNA product to the mature

tRNA form, while the somatic cell extract could

not. Nevertheless, transcription occurred in both

cases.

Schematic representation Schematic representation of two of the zinc fingers in of two of the zinc fingers in TFIIIATFIIIA

Figure 11.46 Schematic representation of two of the zinc ringers in TFIIIA.

The zinc (cyan) in each finger is bound to four amino acids: two cysteines (yellow) and two histidines (blue), holding the finger in the proper shape for DNA binding.

11.3.2 TFIIIB and C11.3.2 TFIIIB and C

Effect of transcription on DNA binding Effect of transcription on DNA binding between a tRNA gene and trranscription between a tRNA gene and trranscription factorsfactors

Binding of TFIIIB and C to Binding of TFIIIB and C to a tRNA genea tRNA gene

Figure 11.48 Binding of TFIIIB and C to a tRNA

gene.

Geiduschek and coworkers performed DNase

footprinting with a labeled tRNA gene (all lanes), and

combinations of purified TFIIIB and C Lane a, negative

control with no factors; lane b, TFIIIC only; lane c,

TFIIlB plus TFIIIC; lane d, TFIIIB plus TFIIIC added,

then heparin added to strip off any loosely bound

protein Note the added protection in the upstream

region afforded by TFIIIB in addition to TFIIIC (lane c)

Note also that this upstream protection provided by

TFIIIB survives heparin treatment, while the protection

of boxes A and B does not Yellow boxes represent

coding regions for mature tRNA Boxes A and B within

these regions are indicated in blue.

Order of binding of Order of binding of transcription factors to a transcription factors to a 5S rRNA gene5S rRNA gene

Figure 11.49 Order of binding of transcription factors to a 5S rRNA gene.

Setzer and Brown added factors TFIIIA, B,

and C, one at a time to a cloned 5S rRNA gene

bound to cellulose. After each addition, they

washed away any unbound factor before

incubation with the next factor. Finally, they

added polymerase Ill and nucleotides, one of

which was labeled, and assayed 5S rRNA

synthesis by electrophoresing the products. The

order of addition of factors is indicated at the

top of each lane. Only when TFIIIB was added

last did accurate 5S rRNA gene transcription

occur. Thus, TFIIIB appears to need the help of

the other factors to bind to the gene.

11.3.3 The Role of TBF11.3.3 The Role of TBF

Figure 11.50 Hypothetical scheme for assembly of the preinitiation complex on a classical polymerase III promoter (tRNA), and start of

transcription.

(a) TFIIIC (light green) binds to the internal promoter’s A and B blocks (green). (b) TFIIIC promoters binding of TFIIIB (yellow), with its TBP (blue) to the region upstream of the transcription start site. (c) TFIIIB promoters polymerase III (red) binding at the start site, ready to begin transcribing. (d) Transcription begins. As the polymerase moves to the right, making RNA (not shown), it presumably removes TFIIIC from the internal promoter. But TFIIIB remains in place, ready to sponsor a new round of polymerase binding and transcription.

Transcription of polymerase Transcription of polymerase III genes complexed only III genes complexed only with TFIIIBwith TFIIIB

Figure 11.51 Transcription of polymerase III genes complexed only with TFIIIB.

Figure 11.51 Transcription of polymerase III genes complexed only with TFIIIB.

Geiduschek and coworkers made complexes containing a tRNA gene and TFIIIB and C (two panels at left), or a 5S rRNA gene and TFIIIA, B, and C (two panels at right), then removed TFIIIC with heparin (lanes e-h), or TFIIIA and C with a high ionic strength butter (lanes l-n). They passed the stripped templates through gel filtration columns to remove any unbound factors, and demonstrated by gel retardation and DNase footpdnting (not shown) that the purified complexes contained only TFIIIB bound to the upstream regions el the respective genes. Next, thsy tested these stripped complexes alongside unstripped complexes for ability to support single-round transcription (S; lanes a, e, i, and l), or multiple-round transcription (M; all other lanes) for the times indicated at bottom. They added extra TFIIIC in lanes c and g, and extra TFIIIB in lanes d and h as indicated at top. They confined transcription to a single round in lanes a, e, i, and I by including a relatively low concentration of heparin, which allowed elongation of RNA to be completed, but then bound up the released polymerase so it could not re-initiate. Notice that the stripped template, containing only TFIIIB, supported just as much transcription as the unstripped template in both single-roued and multiple-round experiments, even when the experimenters added extra TFIIIC (compare lanes c and g, and lanes k and n). The only case in which the unstripped template performed better was in lane d, which was the result of adding extra TFIIIB. This presumably resulted from some remaining free TFIIIC that helped the extra TFIIIB bind, thus allowing more preinitiation complexes to form.

Model of preinitiation complexes Model of preinitiation complexes on TATA-less promoters on TATA-less promoters recognized by all three recognized by all three polymerasespolymerases

Figure 11.52 Model of

preinitiation complexes on TATA-

less promoters recognized by all

three polymerases.

In each case, an assembly factor

(green) binds first (UBF, Spl, and

TFIIIC in class I, II, and III

promoters, respectively), This in turn

attracts another factor (yellow),

which contains TBP (blue); this

second factor is SL1, TFIID, or

TFIIIB in class I, II, or III promoters,

respectively. These complexes are

sufficient to recruit polymerase for

transcription of class I and III

promoters, but in class II promoters

more basal factors (purple) besides

polymerase II must bind before

transcription can begin.