Latchman. Eukariotic Transciption Factors

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present. Unlike TIF-1, SL1 does not exhibit sequence specific binding to the

ribosomal RNA promoter. Hence UBF ac ts by binding to the DNA in a

sequence specific manner and facilitating the binding of SL1. Thus, while

both SL1 and its homologue TIF-1 act as transcription factors necessary for

polymerase I binding, UBF is an additional assembly factor required for bind-

ing of SL1 in vertebrates but not of TIF-1 in Acanthamoeba. This example

therefore illustrates the distinction between factors required only for assembly

of the complex or for binding of the polymerase and transcription itself

(Fig. 3.3).

3.4 RNA POLYMERASE III

The different roles of transcription factors and assembly fact ors are also well

illustrated by the RNA polymerase III system (for reviews see Geiduschek and

Kassavetis, 2001; Paule and White, 2000; Schramm and Hernandez, 2002).

Thus three different classes (I–III) of RNA polymerase III transcription unit

exist, all of which require the essential factor TFIIIB for transcription (for

review see Hernandez, 1993).

In the case of class I transcription units encoding the 5S ribosomal RNAs,

transcription by RNA polymerase III requires the binding of three additional

60 EUKARYOTIC TRANSCRIPTION FACTORS

Figure 3.3

Comparison of ribosomal

RNA gene transcription in

Acanthamoeba and

vertebrates. In

vertebrates, transcription

requires both the TIF-1

homologue SL1 and an

additional assembly factor

UBF whose prior binding

is necessary for

subsequent binding of

SL1.

factors TFIIIA, TFIIIB and TFIIIC. Although both TFIIIA and TFIIIC exhibit

the ability to bind to 5S DNA in a sequence specific manner, TFIIIB like SL1

cannot do so unless TFIIIC has already bound. Once the complex of all these

factors has formed and the RNA polymerase has bound, TFIIIA and TFIIIC

can be removed and transcription continues with only TFIIIB and the poly-

merase bound to the DNA. Hence like UBF, TFIIIA and TFIIIC are assembly

factors which are required for the binding of the tr anscription factor TFIIIB.

In turn, bound TFIIIB is recognized by the polymerase itself and transcription

begins (Fig. 3.4). As with RNA polymerase I, RNA polymerase III binds to the

region of DNA adjacent to that which has bound the transcription factor,

binding of the polymerase being independent of the DNA sequence in this

region.

Although the transcription of the class II RNA polymerase III transcription

units, such as those encoding the tRNAs, is similar to that described for the 5S

RNA genes, TFIIIA is not required. Rather transcription is dependent only

upon TFIIIB and TFIIIC with binding of TFIIIC being sufficient for subse-

quent binding of TFIIIB and the polymerase. Similarly, the class III RNA

polymerase III transcription units, which have a TATA box in the promoter

(for review see Sollner-Webb, 1988) that resembles that found in RNA poly-

RNA POLYMERASES AND THE BASAL TRANSCRIPTIONAL COMPLEX 61

Figure 3.4

Binding of factors to the

5S RNA gene.

Transcription requires the

initial binding of the

assembly factors TFIIIA

and TFIIIC with

subsequent binding of

the transcription factor

TFIIIB and of RNA

polymerase III itself.

merase II promoters (see Chapter 1, section 1.3.2) also require TFIIIB for

transcription together with other accessory factors (for discussion see

Hernandez, 1993).

The process of transcription by RNA polymerases I and III therefore

involves the binding of a single transcription factor to the promot er, allowing

subsequent binding of the RNA polymerase to an adjacent region of DNA.

The transcription factor remains bound at the promoter as the polymerase

moves down the DNA allowing repeated binding of polymerase molecules

and hence repeated rounds of transcription. Binding of the polymerase to

the promoter requires prior binding of the transcription factor since the

polymerase does not recognize a specific sequence in the promoter but rather

makes protein–protein contact with the transcription factor and binds to the

adjacent reg ion of the DNA.

In different syst ems, however, different requirements exist for the binding

of the transcription factor itself. Thus in the Acanthamoeba system, TIF-1 can

bind to DNA in a sequence specific manner and hence is the only factor

required. In most other systems, this is not the case and the transcription

factors do not bind to the DNA unless other assembly factors, which exhibit

sequence specific DNA binding, are present. Once the transcription factor has

bound, these assembly factors can be removed, for example, by detergent

treatment without affecting subsequent transcription. It is unclear, however,

whether these factors do actually dissociate from the complex under normal

conditions in vivo once the transcription factor has bound (for discussion see

Paule, 1990). Whatever the case, the transcription factor itself remains bound

at the promoter even after the polymerase has moved down the gene, allowing

repeated binding of polymerase molecules and hence repeated rounds of

transcription.

Although assembly factors play only an accessory role in transcription

itself, they are essential if the complex is to assemble. Hence both assembly

factors and transcription factors can be the target for processes which reg-

ulate the rate of transcription (for review see Brown et al., 2000). Thus, while

the high rate of polymerase III transcription in embryonal carcinoma cells is

dependent on a high level of transcription factor TFIIIB, the increase in

transcription by this polymerase following adenovirus infection is due to an

increase in the activity of the assembly factor TFIIIC. Similarly, alterations in

the level of TFIIIA during Xenopus development control the nature of the 5S

rRNA genes that are transcribed at different developmental stages. In

addition, as will be discussed in Chapter 9 (section 9.4.3) the retinoblastoma

anti-oncoprotein inhibits cellular gr owth by interacting with UBF to inhibit

RNA polymerase I activity and with TFIIIB to inhibit RNA polymerase III

activity.

62 EUKARYOTIC TRANSCRIPTION FACTORS

RNA POLYMERASES AND THE BASAL TRANSCRIPTIONAL COMPLEX 63

3.5 RNA POLYMERASE II

3.5.1 STEPWISE ASSEMBLY OF THE RNA POLYMERASE II

BASAL TRANSCRIPTIONAL COMPLEX

Although some regulation of RNA polymerase I and III activity does occur

therefore, this is much less extensive compared to the very wide variety of

regulatory events affecting the activity of genes transcribed by RNA poly-

merase II. As discussed above, this results in a bewildering array of tran-

scription factors interacting with this enzyme and conferring particul ar

patterns of regulation. Interestingly, however, even the basal transcriptional

complex, which is essential for any transcription by this enzyme, contai ns fa r

more components than is the case for the other RNA polymerases (for

reviews see Orphanides et al., 1996; Roeder, 1996; Woychick and

Hampsey, 2002).

One component of this complex which has been intensively studied and

plays an essential role in RNA polymerase II mediated transcription is TFIID

(for review see Burley and Roeder, 1996). In promoters containing a TATA

box (see Chapter 1, section 1.3.2), TFIID binds to this element, protecting a

region from thirty-five bases to nineteen bases upstream of the start site of

transcription in the human hsp70 promoter, for example. The binding of

TFIID to the TATA box or equivalent region is the earliest step in the forma-

tion of the stable transcriptional complex, such binding being facilitated by

another factor TFIIA (Fig. 3.5a).

Interestingly, as TFIID is progressively purified, its requirement for TFIIA

to aid its activity decreases. This is because in less purified preparations and in

the intact cell, TFIID is associated with a number of inhibitory factors such as

Dr1 and Dr2 (for review see Drapkin et al., 1993) which act by preventing its

binding to the DNA and/or its interaction with other components of the basal

complex such as TFIIB (see below) (for further discussion of the role of Dr1,

see Chapter 6, section 6.3.3). One role of TFIIA appears to be to bind to

TFIID and overcome this inhibition, ther eby stimulating the activity of TFIID.

Hence the need for TFIIA decreases as TFIID is purified away from these

inhibitory factors, although it is likely to play a critical role in the intact cell. In

addition, TFIIA may also play a role in the response to transcriptional activa-

tors acting as a co-activator molecule linking DNA-bound activators and the

basal transcriptional complex.

Hence rather than acting as a basal transcription factor essential for all

transcription, TFIIA appears to play a key role in the response of the complex

to activating and inhibiting molecules. Such a role is of particular importance

since the antagonism between positively and negatively acting factors in the

assembly of the basal transcriptional complex may play a critical role in reg -

ulating the rate of transcription, representing a major target for activators and

repressors of transcription (see Chapters 5 and 6, for a further discussion of

the mechanisms by which specific factors activate or inhibit transcription).

Once TFIID has bound to the DNA, another transcription factor TFIIB,

joins the complex by binding to TFIID (Fig. 3.5b). This binding of TFIIB is an

essential step in initiation complex formation since, as well as binding to

TFIID, TFIIB can also bind to the RNA polymerase itself. Hence it acts as a

64 EUKARYOTIC TRANSCRIPTION FACTORS

Figure 3.5

Stages in the assembly

of the stable

transcriptional complex for

RNA polymerase II

transcription. As the

polymerase moves away

from the promoter to

transcribe the gene, TFIIF

remains associated with it

while TFIIA and TFIID

remain bound at the

TATA box allowing the

formation of a new stable

complex and further

rounds of transcription.

bridging factor allowing the recruitment of RNA polymerase to the complex

in association with another factor TFIIF (Fig. 3.5c). Following polymerase

binding, three other transcription factors TFIIE, TFIIH and TFIIJ, rapidly

associate with the complex (Fig. 3.5d). At this point, TFIIH, which has a

DNA helicase activity, unwinds the double-stranded DNA so allowing it to

be copied into RNA. Subsequently, the kinase activity of TFIIH, which allows

it to phosphorylate other proteins, phosphorylates the C-terminal domain of

RNA polymerase (for review see Orphanides et al., 1996). This converts it

from the non-phosphorylated form which joins the complex to the phosphory-

lated form which is capable of transcriptional elongation to produce the RNA

product (Fig. 3.6) (see section 3.1).

Hence TFIIH, via its kinase and helicase activities, plays a critical role in

allowing the basal transcriptional complex to initiate transcription. Moreover,

TFIIH also plays a critical role in the repair of damaged DNA, providing a

possible link between the processes of DNA repair and transcription (f or

reviews of TFIIH see Hoeijmakers et al., 1996; Svejstrup et al., 1996).

Interestingly, it has recently been shown that the kinase activity associated

with TFIIH can also phosphorylate the retinoic acid receptor which is a mem-

RNA POLYMERASES AND THE BASAL TRANSCRIPTIONAL COMPLEX 65

Figure 3.6

TFIIH has a helicase

activity which unwinds

the DNA allowing its

transcription into RNA

and a kinase activity that

phosphorylates the C-

terminal region of RNA

polymerase which allows

it to begin transcription.

ber of the nuclear receptor transcription factor family discussed in Chapter 4

(section 4.4). This phosphorylation stimulates the ability of the retinoic acid

receptor to activate transcription (Rochette-Egly et al., 1997) indicating that

TFIIH may play a role in the regulation of transcription factor activity by

phosphorylation (see Chapter 8, section 8.4.2).

The complex of the seven factors (TFIIA, B, D, E, F, H and J) and the

polymerase is thus sufficient for transcription to occur. As the polymerase

moves down the gene during this process TFIIF remains associated with it

while TFIIA and TFIID remain bound at the promoter and are capable of

binding another molecule of polymerase allowing repeated rounds of tran-

scription as with the other polymerases (see Fig. 3.5e).

3.5.2 THE RNA POLYMERASE HOLOENZYME

Although the step-by-step pathway of assembling the basal transcriptional

complex described above was proposed on the basis of a number of studies,

an alternative pathway has also be en identified base d on the finding that some

RNA polymerase is found in solution already associated with TFIIB, TFIIF

and TFIIH in the absence of DNA. This so-called RNA polymerase holoen-

zyme has now been observed in a wide range of organisms ranging from yeast

to man. It is clear therefore that, in some cases, following binding of TFIIA

and TFIID to the promoter, this complex of RNA polymerase and associated

factors may bind resulting in a reduced number of steps being required for

complex formation (Fig. 3.7) (for discussion see Pugh, 1996; Greenblatt, 1997;

Myer and Young, 1998).

Interestingly, the RNA polymerase holoenzyme also contains a number of

other components apart from RNA polymerase itself and the basal transcrip-

tion factors. Thus it includes a complex of proteins, known as the mediator

complex, which appears to be required, at least in yeast, for the response to

transcriptional activators (see Chapter 5, section 5.4.1). Hence the mediator

may serve as a link between these activators and the components of the basal

transcriptional complex whose activity they stimulate. In addition, the holoen-

zyme can also associate with the SWI/SNF complex discussed in Chapter 1

(section 1.2.2) whose role is to remodel the chromatin into a form whi ch

allows the binding of transcriptional activators and transcription itself.

Hence, at least in some cases, this remodelling complex can be recruited to

DNA together with the RNA polymerase and its associated proteins.

The RNA polymerase holoenzyme is thus a highly complex structure which,

as well as RNA polymerase itself and basal transcription factors, also contains

factors involved in the response to transcriptional activators and others which

remodel chr omatin structure. Altho ugh this holoenzyme represents only one

66 EUKARYOTIC TRANSCRIPTION FACTORS

of the two possible methods by which the basal transcription complex assem-

bles on the DNA, it is clear that regardless of its method of assembly the basic

stable transcriptional complex for RNA polymerase II requires a number

of factors in addition to the polymerase itself and is therefore much more

complex than that of RNA polymerase I or III.

3.6 TBP, THE UNIVERSAL TRANSCRIPTION FACTOR?

Most of the transcription factors described in the previous sections were

isolated by the biochemical fractionation of cellular extracts and were then

shown to have a particular functional activity in modulating the rate of tran-

scription when mixed with RNA polymerase and other subcellular fractions.

When these factors were characterized in more detail by further fractionation

and subsequent cloning, however, many of them were shown to consist of

several different proteins which together are responsible for the properties

ascribed to the original factor. Thus, although these factors have been dealt

RNA POLYMERASES AND THE BASAL TRANSCRIPTIONAL COMPLEX 67

Figure 3.7

Alternative pathways in

the assembly of the

stable transcriptional

complex for RNA

polymerase II involving

either the step by step

pathway (see Fig. 3.5) or

the binding of a pre-

formed complex of RNA

polymerase and its

associated factors to

DNA which has already

bound TFIIA and TFIID.

with for simplicity in the previous sections as single factors, most of them are

in fact complexes of several different proteins, for example, TFIIE and TFIIF

both contain two distinct protein s. Similarly, TFIIH is a multi-protein complex

whose structure has been determi ned (Chang and Kornbeg, 2000; Schultz et

al., 2000) with one of the component proteins having the kinase activity which

results in phosphorylation of the RNA polymerase while another has the heli-

case activity which unwinds the DNA (see section 3.5.1) (for review see

Hoeijmakers et al., 1996; Svejstrup et al., 1996).

This responsibility of one component of the complex for an activity for-

merly ascribed to the whole complex is seen most clearly in TFIID. Thus,

TFIID is a multi-protein complex in which only one protein known as TBP

(TATA-binding protein) directs the binding to the TATA box while the other

components of the complex, known as TAFs (TBP-associated factors), do not

bind di rectly to the TATA box and appear to allow TFIID to respond to

stimulation by transcriptional activators (see Chapter 5, section 5.4.2)

(for review see Hahn , 1998; Green, 2000). They thus represent co-activator

molecules, linking transc riptional activators and the basal transcriptional

complex.

Hence TBP plays a critical role in the transcription of TATA box-contain-

ing RNA polymerase II promoters by binding to the TATA box as the first

step in assembly of the basal transcriptional complex. In view of this critical

role, it is not surprising that TBP is one of the most highly conserved eukary-

otic proteins. The structure of this protein has been defined by X-ray crystal-

lography and shown to have a saddle structure in which the concave underside

binds to DNA and the convex outer surface is acces sible for interactions with

other factors. Most interestingly, binding of TBP to the DNA deforms the

DNA so that it follows the concave curve of the saddle (Fig. 3.8). Moreover,

structural studies of the TFIID complex (consisting of TBP and the TAFs)

bound to DNA have indicated that it res embles the complex of the eight

histone molecules around which DNA is wound in the nucleosome to form

the normal chromatin structure (see Chapter 1, section 1.2.1). Hence the

DNA may bend around TFIID at the promoter in a similar manner to the

folding of the rest of DNA in the bas ic nucleosome structure of chromatin (for

reviews see Hoffman n et al., 1997; Gangloff et al., 2001). This role for TFIID in

altering nucleosome structure at the promoter is also supported by the find-

ing that TAFII

250

, one of the subunits of TFIID, has histone acetyltransferase

activity (Mizzen et al., 1996), since acetylation of histones appears to play a key

role in modulating chromatin structure (see Chapter 1, section 1.2.3).

The bent DNA with TFIID bound to it serves as the central platform on

which the basal transc riptional complex assembles. Thus, structural studies

have shown that TFIIA binds to the amino terminal stirrup of the TBP saddle

68 EUKARYOTIC TRANSCRIPTION FACTORS

and interacts only with the DNA upstream of the TATA box. This allows it to

fulfil its role of protecting TFIID from inhibition by transcriptional repressors

and allowing it to respond to activators bound to upstream DNA sequences

(see section 3.5.1). In contrast, TFIIB binds to the carboxyl-terminal stirrup of

the TBP saddle and binds to the DNA downstream (as well as upstream) of the

TATA box (Andel et al., 1999). This allows it to fulfill its role of acting as a

bridge between TBP and RNA polymerase II so positioning the start site of

transcription by the polymerase relative to the TATA box (see Pl ate 1; Geiger

et al., 1996) (for reviews see Roeder, 1996; Nikolov and Burley, 1997;

Woychick and Hampsey, 2002). Interestingly, a recent study indicates that

binding of TFIIB promotes bending of the DNA by TBP, indicating that

TFIIB acts by interacting with the partially assembled complex as well as by

recruiting new factors to the complex (Zhao and Herr, 2002).

Paradoxically, in view of its TATA box binding ability, TBP also plays a

critical role in the transcription of the subset of RNA polymerase II genes

which do not contain a TATA box (see Chapter 1, section 1.3.2). In this case,

however, TBP does not bind to the DNA but is recruited to the promoter by

another DNA binding protein which binds to the initiator element overlap-

ping the transc riptional start site. TBP then binds to this initiator binding

protein allowing the recruitment of TFIIB and the RNA polymerase itself as

for promoters containing a TATA box. Hence TBP plays a critical role in the

assembly of the transcription complex for RNA polymerase II, although it

joins the complex by binding to DNA in the case of TATA-box-containing

promoters (Fig. 3.9a) and is recruited by protein–protein interactions in the

case of promoters which lack a TATA-box (Fig. 3.9b).

RNA POLYMERASES AND THE BASAL TRANSCRIPTIONAL COMPLEX 69

Figure 3.8

The saddle structure of

TBP as determined by X-

ray crystallography allows

the concave surface to

interact with the TATA

box while the convex

surface associates with

other accessory

transcription factors (X

and Y). The initial binding

induces the bending of

the DNA so that it

follows the concave

under surface of the

saddle. See also colour

plate 1.