General Transcription Factors (TFs)

An overview

DNA
virus
dNTPs

Nucleolus 4 RNA
Replication
virus

1 Transcription
rRNA DNA

rNTPs

Nucleus
Cytosol
2 RNA A
processing AA
AA

mRNA
Ribosomal Protein
subunits Amino acids
A AA
Translation AA
factors

tRNA

3 mRNA translation

FIGURE 51 Overview of four basic molecular genetic processes. During translation 3 , the four-base code of the mRNA is decoded into
In this chapter, we cover the three processes that lead to production of the 20–amino acid language of proteins. Ribosomes, the macromo-
proteins 1 – 3 and the process for replicating DNA 4 . Because viruses lecular machines that translate the mRNA code, are composed of two
utilize host-cell machinery, they have been important models for study- subunits assembled in the nucleolus from ribosomal RNAs (rRNAs) and
Transcription factors in Eukaryotes
General Transcription Factors
Ø Initiation of transcription by RNA polymerase II requires several initiation factors.
Ø These initiation factors position Pol II molecules at transcription start sites and help
to separate the DNA strands so that the template strand can enter the active site of
the enzyme.
Ø They are called general transcription factors because they are required at most, if
not all, promoters of genes transcribed by RNA polymerase II.
Ø These proteins are designated TFIIA, TFIIB, and so on, and most are multimeric
proteins.
Ø Among them, The largest is TFIID, which consists of a single 38-kDa TATA box–
binding protein (TBP) and 13 TBP-associated factors (TAFs).
Ø General transcription factors with similar activities and homologous sequences are
found in all eukaryotes.
Transcription factors in Eukaryotes
General Transcription Factors
Ø The complex of Pol II and its general transcription factors bound to a promoter and
ready to initiate transcription is called a preinitiation complex (PIC).
Ø I will try to summarizes the current model for the stepwise assembly of the Pol II
transcription preinitiation complex on a promoter containing a TATA box in next
couple of slides.

TAFs
Unbound promoter
TFIID
TBP

Promoter DNA TATA

box
TFIIA
TFIIB
 encoded mRNA.
Transcription factors in Eukaryotes
seGeneral Transcription
II Promoters and Factors
The TATA Box, Initiators, and CpG Islands
Function as Promoters in Eukaryotic DNA
onØFactors
The TBP subunit of TFIID is the first
Several different types protein to bindcan
of DNA sequences tofunction
a TATA as box promoter.
late transcription initiation and promoters for RNA polymerase II, telling the polymerase
eraseØ All eukaryotic TBPs where
II have been studied exten- analyzed to transcription
to initiate date haveofvery an RNA similar C-terminal domains of 180
complementary
rase is the one that transcribes to the template strand of a double-stranded DNA molecule.
tiation and elongation by RNA
residues.
biochemical processes required
These sequences include TATA boxes, initiators, and CpG
islands.
n-coding genes and are the steps
mostØ frequently
This domainregulated toofde-TBPTATA
folds
Boxesinto a saddle-shaped
The first genes to be sequencedstructure;
and studied the two halves of the
h cells specific proteins are syn- through in vitro transcription systems were viral genes and
previous molecule
section, theexhibit
expressionan overall dyad symmetry
cellular protein-coding butarearevery
genes that not identical.
actively tran-
ng genes is regulated by multiple scribed, either at particular times of the cell cycle or in spe-
Ø TBP
ences, interacts
generically referred with
to as the minor
cific groove
differentiated cell in DNA,
types. bending
In all these highly the helix considerably
transcribed
ns. These sequences include pro- genes, a conserved sequence called the TATA box was found
where transcription of the DNA about 26–31 bp upstream of the transcription start site
Ø The DNA-binding
types of control elements located surface of TBP is conserved in all eukaryotes, explaining the
(Figure 9-16). Mutagenesis studies have shown that a single-
, as well as sequences located far base change in this nucleotide sequence drastically decreases
, calledhigh conservation
enhancers, which control of the TATA box promoter element
he gene is transcribed and how
In this section, we take a closer
∼ −37 to −32 ∼ −31 to −26 −2 to + 4 + 28 to + 32
arious transcription-control ele-
protein-coding genes and some
them.

ates Transcription at BRE TATA box Inr

Initiator
DPE
Downstream
TFIIB
sponding to the 5′ Cap of recognition Drosophila + 1 G T promoter element
element A AA TCA T
TATA A T C G
GGG T TG T A AC
CGCC G A
C CA Mammals YYAN YY G TT
periments using purified RNA A C
xtract prepared from the nuclei FIGURE 916 Core promoter elements of non-CpG island pro-
Transcription factors in Eukaryotes
General Transcription Factors
Ø Once TFIID has bound to the TATA box, TFIIA and TFIIB can bind.
Ø TFIIA is a heterodimer larger than TBP, and TFIIB is a monomeric protein, slightly
smaller than TBP.
Ø TFIIA associates with TBP and DNA on the upstream side of the TBP–TATA box
complex.
TAFs
Ø The C-terminal domain of TFIIB makes contact with
Unbound promoter both TBP and DNA on either
TFIID
side of the TATA box. TBP

Ø During transcription initiation, its N-terminal domain is inserted

Promoter DNA TATA
into the RNA exit
box
channel of RNA polymerase II TFIIA
TFIIB

Upstream promoter +1
complex
Transcription factors in Eukaryotes
General Transcription Factors
Ø During transcription initiation, its N-terminal domain (TFIIB) is inserted into the
RNA exit channel of RNA polymerase II.
Ø The TFIIB N-terminal domain assists Pol II in melting the DNA strands at the
transcription start site and interacts with the template strand near the Pol II active
site.
Ø Following TFIIB binding, a preformed complex of TFIIF (a heterodimer of two
different subunits in mammals) and Pol II binds, positioning the polymerase over
the start site.
Ø Two more general transcription factors must bind before the DNA duplex can be
separated to expose the template strand.
Ø First to bind is TFIIE, a heterodimer of two different subunits.
Ø TFIIE creates a docking site for TFIIH (another multimeric factor containing 10
different subunits). Binding of TFIIH completes assembly of the transcription
preinitiation complex
 box nas
polymerase. The DNA is held in position in the PIC by binding of the
Transcription factors in Eukaryotes
TFIIB
TFIIA
structure of the duplex DNA assists ATP
mul
TATA box by the TBP subunit of TFIID, and the resulting strain on the
Thr
the N-terminal region of TFIIB and
General Transcription Factors Pol II to melt the DNA at the transcription start site, forming the tran-
will
scription bubble. As Pol II initiates transcription in the resulting open
late
Ø TFIIE creates
Upstream promoter a docking
+1 site for TFIIH (another multimeric factor containing 10
complex, the polymerase transcribes away from the promoter, its CTD
cap
becomes phosphorylated by the TFIIH kinase domain, and the general
different subunits). Binding of TFIIH completes assembly of the transcription
complex
Open PIC
transcription tran
factors dissociate from the promoter. See S. Sainsbury,
tran
C. Berrnecky, and P. Cramer, 2015, Nat. Rev. Mol. Cell Biol. 16:129.
preinitiation complex com
CTD
Transcription bubble bou
Pol II awa
TFIIF NTPs
of 1.5 megadaltons (MDa)—about the size of a ribosomal tran
subunit. Nascent RNA
Such elaborate preinitiation complexes assemble at
the promoters of every protein-coding gene expressed by a
Core PIC
eukaryotic
Initially cell.
transcribing
complex The helicase activity of one of the core TFIIH subunits them
Upstream Downstream (Ssl2 in yeast; see Figure 9-20d) uses energy from ATP bas
DNA DNA hydrolysis to help unwind the DNA duplex at the start min
TFIIE site, allowing Pol II to form an open complex in which whe
the DNA duplex surrounding the startfactors
Elongation site is melted and regi
TFIIH TFIIH the template strand is bound at the polymerase active site.
kinase Initiation factors P
P
P
lack
As the polymerase transcribes away P P from
P the promoter re- 9-1
gion, the N-terminal
Elongation 5’ cap domain Pof PTFIIB is released from the reco
RNA exit channel as the 5′ end of the nascent RNA enters
complex
Closed PIC oth
it. Three TFIIH subunits form a kinase module (TFIIH ki-
nase in Figure 9-19) that phosphorylates the Pol II CTD
dam
multiple times on serine 5 (underlined) of the Tyr-Ser-Pro- the
ATP Thr-Ser-Pro-Ser repeat that constitutes the CTD. As we
will discuss further in Chapter 10, a multiply phosphory-
9.3 RNA Pol
lated CTD is a docking site for the enzymes that form the
cap structure (see Figure 5-14) on the 5′ end of an RNA
Open PIC transcribed by RNA polymerase II. In the minimal in vitro
transcription assay with TBP substituted for the full TFIID
complex and purified RNA polymerase II, TBP remains
Transcription bubble bound to the TATA box as the polymerase transcribes
Transcription factors in Eukaryotes
General Transcription Factors
Ø To date, researcher has completely explored the cryoelectron microscopic image of a
yeast (S. cerevisiae) preinitiation complex assembled in vitro from purified RNA
polymerase II and general transcription factors with TBP in place of the complete
TFIID complex.
Ø A total of thirty-three polypeptides with a mass of 1.5 megadaltons (MDa), about the
size of a ribosomal subunit.
Ø Such elaborate preinitiation complexes assemble at the promoters of every protein-
coding gene expressed by a eukaryotic cell.
Ø In next slide I am going to show you the “detailed model of the yeast preinitiation
complex based on cryoelectron microscopy and fitting of known protein x-ray
crystal structures”.
Transcription factors in Eukaryotes
General Transcription Factors
Ø Model of the yeast preinitiation complex based on cryoelectron microscopy and
fitting of known protein x-ray crystal structures. (a-c) Three views of the nearly
complete PIC.

(a) Side (b) Front (c) Back

TFIIH
TFIIH TFIIH
90° 180°
Pol II
TFIIE TFIIB TFIIE Pol II
TFIIE
Clamp Ssl2
Clamp Ssl2
Ssl2 m
strea
TBP Down
TFIIH TBP DNA

TFIIB TFIIF TFIIF TFIIH

TFIIA TFIIS
TFIIA TFIIF TFIIS
TFIIF
Upstream
Upstream
DNA
DNA

(d)
TFIIA TFIIA
Ssl2 Ssl2
TBP TBP

ATP
TFIIB TFIIB
Transcription factors in Eukaryotes
General Transcription Factors
Ø Now Specifically, we will talk about the function of TFIIH (and its helicase subunit)
Ø The helicase activity of one of the core TFIIH subunits (Ssl2 in yeast) uses energy
from ATP hydrolysis to help unwind the DNA duplex at the start site, allowing Pol II
to form an open complex in which the DNA duplex surrounding the start site is
melted and the template strand is bound at the polymerase active site.
Ø As the polymerase transcribes away from the promoter region, the N-terminal
domain of TFIIB is released from the RNA exit channel as the 5′ end of the nascent
RNA enters it.
Ø Three TFIIH subunits form a kinase module (TFIIH kinase as I mention in previous
slide) that phosphorylates the Pol II CTD multiple times on serine 5 of the Tyr-Ser-
Pro-Thr-Ser-Pro-Ser repeat that constitutes the CTD.
Transcription factors in Eukaryotes
General Transcription Factors
Ø This multiple phosphorylated CTD is a docking site for the enzymes that form the
cap structure (We will discuss this point in RNA processing) on the 5′ end of an
RNA transcribed by RNA polymerase II.
Ø In the minimal in vitro transcription assay with TBP substituted for the full TFIID
complex and purified RNA polymerase II, TBP remains bound to the TATA box as
the polymerase transcribes away from the promoter region, but the other general
transcription factors dissociate
 To be continued
See you tomorrow IA