Transcript Genomes 3/e - Illinois Institute of Technology

Chapter 11:
Transcription Initiation
Complex
Copyright © Garland Science 2007
Genome expression includes 2 steps
• Initiation of transcription. Assembly of
upstream protein complex (e.g. RNA
polymerase & accessory proteins)
This step determines whether a gene
should be expressed or not.
• Synthesis & processing of RNA (next
Chapter). RNA polymerase synthesizes
mRNA & subsequently processes or
modifies into mature mRNA.
Figure 11.1 Genomes 3 (© Garland Science 2007)
11-1. DNA
binding
proteins are
the key to
initiate
transcription.
DNA binding
proteins play a wide
variety of functions
(e.g. RNA
transcription, DNA
replication, repair,
recombination, etc.)
Table 11.1 Genomes 3 (© Garland Science 2007)
11-1-1. (Cont.)
Proteins contain highly
specific regions in direct
contact w/DNA (called
“DNA binding motifs”).
Helix-turn-helix motif
(20 AA in length) common
in both pro- & eukaryotes;
consists of two α-helix
units & one β-turn; 2nd αhelix recognizes/contacts
DNA major groove; in
bacteria: lactose repressor;
in eukaryotes: POU domain
& winged HTH motif.
Figure 11.2 Genomes 3 (© Garland Science 2007)
11-1-1. (Cont.)
Zinc fingers common in
eukaryotes; 1% mammalian
genes encode zinc fingers; 6
types; well-studied Cys2His2
finger consists of one αhelix units & one β-sheet;
α-helix recognizes/contacts
DNA major groove; Zinc
atom to stabilize the finger
structure; a single protein
sometimes contains multiple
copies of zinc fingers.
Figure 11.4 Genomes 3 (© Garland Science 2007)
11-1-1. (Cont.)
Ribbon-helix-helix
motif common in
bacteria; the ribbon (βsheets) contacts DNA
major groove.
TATA binding protein
(or TBP domain) contacts
minor groove of DNA.
RNA binding proteins
include RNP domain,
dsRNA binding domain, κhomology domain, etc.
Figure 11.6 Genomes 3 (© Garland Science 2007)
11-1-2. DNA-binding sites in a genome
Attachment sites for DNA-binding proteins are
usually located immediately upstream of a gene;
help to identify real genes in a genome (e.g. to
search several Kb upstream).
Figure 11.7 Genomes 3 (© Garland Science 2007)
11-1-2. (Cont.)
Identification of
DNA binding
protein by
experimental
techniques
Gel retardation
Restriction collection
mixed with nuclear
proteins; DNA-protein
complex impede gel
electrophoresis.
Figure 11.8 Genomes 3 (© Garland Science 2007)
11-1-2. (Cont.)
Modification
protection assay 1
Restriction fragments
end labeled; mixed
with nuclear proteins;
add nuclease under
limiting conditions to
make 1 random cut
per fragment; DNAprotein complex will
not be digested;
compare gel
electrophoresis.
Figure 11.9 Genomes 3 (© Garland Science 2007)
11-1-2. (Cont.)
Modification
protection assay 2
Restriction fragments
end labeled; mixed
with nuclear proteins;
add DMS under
limiting conditions to
methylate 1 guanine
per fragment; DNAprotein complex will
not be methylated;
compare gel
electrophoresis.
Figure 11.10 Genomes 3 (© Garland Science 2007)
11-1-3. DNA
sequence influence
DNA binding
protein
Configuration effect
B-form or Z-form: major
groove can be “direct
readout”. A-form is
difficult to read.
DNA bending
repeated adenines cause
bending at the 3’ end.
Figure 11.12 Genomes 3 (© Garland Science 2007)
11-1-4. Interaction
between DNA & DNA
binding protein
Most are electrostatic & noncovalent (between - charges of
DNA & + charges of protein R
groups)
Recognize specific
(thermodynamically favorable)
DNA sequences but can also
bind non-specifically; dimers
structure maximizes interaction
w/major groove.
Figure 11.13 Genomes 3 (© Garland Science 2007)
11-2. DNA-protein interactions to
initiate transcription.
11-2-1. DNA-dependent RNA polymerase
In eukaryotes: 3 distinct types (I for rRNAs, II for
proteins, III for tRNAs) consisting of 8-12 subunits
In bacteria: RNA polymerase consists of α2ββ’σ
subunits
Table 11.3 Genomes 3 (© Garland Science 2007)
11-2-1. (Cont.)
In bacteria: RNA
polymerase directly
attach promoters
(where RNA
polymerase binds
upstream of genes)
In eukaryotes: DNAbinding proteins first
bind & then RNA
polymerase binds
Figure 11.14 Genomes 3 (© Garland Science 2007)
11-2-1. (Cont.)
E. coli promoter contains two 6-nt segments:
-35 box 5’-TTGACA-3’
-10 box 5’-TATAAT-3’
+1 is where transcription begins
20-600 nt upstream of the start codon
Figure 11.15 Genomes 3 (© Garland Science 2007)
11-2-1. (Cont.)
E. coli promoter position is relatively conserved;
-35 & -10 box sequences can vary from genes to
genes (see below); but mutation in promoter
regions prevents gene expression.
Table 11.4 Genomes 3 (© Garland Science 2007)
11-2-1. (Cont.)
Eukaryotic promoter is where initiation complex is
assembled; usually consists of Core promoter +
upstream promoter elements. e.g. RNA polymerase
II has a core promoter (TATA box + initiator
sequence) plus downstream promoter element, GCrich motif, proximal sequence element.
Figure 11.16 Genomes 3 (© Garland Science 2007)
11-2-2. Assembly
of transcription
initiation complex
General steps:
1. Attach to promoter
sequences;
2. Convert from a closed
complex to an open
complex;
3. Move away from
promoter & initiate
transcription.
Figure 11.17 Genomes 3 (© Garland Science 2007)
11-2-2. (Cont.)
In E. coli:
Attach to promoter
sequences is specified
by σ subunit & -35 box;
Convert from a closed
complex to an open
complex is based on -10
box; σ subunit
dissociates soon after
transcription initiates.
Figure 11.18 Genomes 3 (© Garland Science 2007)
11-2-2. (Cont.)
In eukaryotes:
The process is similar but
RNA polymerase II does
not directly recognize
promoter sequences;
instead, general
transcription factor (GTF)
binds to DNA; “saddlelike” structure.
Figure 11.19 Genomes 3 (© Garland Science 2007)
11-3. Regulation
of transcription
initiation
Primary regulation occurs
at the level of
transcription initiation &
decides which gene is
expressed in a particular
cell & relative rate
Secondary regulation is
during the posttranscription (e.g. mRNA
modification) and the
protein synthesis &
modification.
Figure 11.22 Genomes 3 (© Garland Science 2007)
11-3. (Cont.)
Two levels of regulation:
Constitutive control by
promoter structure (basal
level of transcription)
Regulatory control by
regulatory proteins
(transcription initiation).
In E. coli, -35 box
influences σ subunit
recognition & RNA
polymerase attachment;
strong promoters direct
x1000 more productive
initiations than weak
promoters.
Figure 11.23 Genomes 3 (© Garland Science 2007)
11-3. (Cont.)
Regulatory control in E. coli, the concept of
operator (a region between promoter and operon
& regulates the initiation of operon).
A few transcripts
Figure 11.24 Genomes 3 (© Garland Science 2007)
5000 transcripts
11-3. (Cont.)
Regulatory control in E. coli, tryptophan (the gene
product itself) is a co-repressor to inactivate
operon expression. The process is called
“feedback inhibition”.
Figure 11.25 Genomes 3 (© Garland Science 2007)
11-3. (Cont.)
In addition to repressors,
there are activators
which increase the
efficiency of transcription
initiation;
Same proteins bind more
than 1 promoters (see
left);
Recognition sequences
can be enhancers or
silencers by
conformational changes.
Figure 11.26 Genomes 3 (© Garland Science 2007)
11-3. Regulation of transcription
initiation in eukaryotes
RNA polymerase II promoter consists of many short
sequence regions, including core promoter (TATA
box & Inr sequence), basal promoter elements
(CAAT box, GC box, etc), response modules, cellspecific modules, developmental regulators.
Figure 11.27 Genomes 3 (© Garland Science 2007)
11-3. Regulation of transcription
initiation in eukaryotes (Cont.)
Alternative promoters also contribute to the
transcription regulation, e.g. human dystrophin gene
(the largest gene spanning 2.4 Mb w/78 introns) has
>7 tissue-specific alternative promoters (e.g.
cortical tissue, muscles, cerebellum, etc.)
Figure 11.28 Genomes 3 (© Garland Science 2007)
11-3. Regulation of transcription
initiation in eukaryotes (Cont.)
Activators & co-activators bind to upstream
promoter elements & enhancers (activation
domain); interact with RNA polymerase II & regulate
a single gene or multiple genes.
Figure 11.29 Genomes 3 (© Garland Science 2007)
11-3. (Cont.)
Activators & co-activators
interact with RNA
polymerase II via another
protein complex called
mediator. (Left) yeast
mediator, detailed
mechanism is not clear.
Repressors are also
important in eukaryotes,
e.g. inhibit assembly of preinitiation complex; activators
& repressors themselves are
controlled by synthesis &
conformational changes.
Figure 11.30 Genomes 3 (© Garland Science 2007)
Chapter 11 Summary
DNA-binding proteins play a central role in
transcription; many can attach to specific DNA
sequences (e.g. helix-turn-helix or zinc finger);
some can directly read DNA sequences in major
grooves which can be affected by DNA
conformation.
Promoters specify where transcription initiation
complex should be assembled; bacteria have a
single RNA polymerase which directly attach to 2
promoter regions; eukaryotes have 3 RNA
polymerases & more complex promoters which
interact via general transcription factors;
activators & repressors can further regulate
transcription initiation.