Tetrameric
Structure of IRF-3 with DNA
Andy Schwartz '12 and Luke Sullivan '12
Contents:
I.
Overview
The recruitment and
binding of transcription factors to enhancers is crucial for mammals.
When mammals are infected with a virus, the β-interferon gene
is
activated through the help of transcription factors. There are several
activators that bind to the enhancer region of β-interferon
between
-110 and -45 nucleotides away from the promoter. This region of DNA
contains four overlapping positive regulatory domains that are bound by
transcriptional proteins. Together, these proteins form an enhanceosome,
which is responsible for the recruitment of
chromatin modifying and other transcription factors. The enhanceosome
is made through the help of HMGA1, an architectural protein. HMGA1
binds the minor groove of DNA, straightening the bent enhancer region.
It also assists in recruiting the activators that compose the
enhanceosome- NF-κB, Jun/ATF, and IRF. There 9 different
mammalian
IRFs, but research has shown that IRF-3 and IRF-7 are especially
important in the expression of the β-interferon gene. When a
virus
attacks, the IRF-3 molecules found in tissues are turned on by the
phosphorylation of serine residues. IRF-3 then forms a dimer, moves
from the cytoplasm to the nucleus, and binds to the PRDIII and PRDI
elements of the enhancer via recognition helices
.
II.
General Structure
Four IRF-3 molecules,
IRF-3A
, IRF-3B,
IRF-3C
and IRF-3D,
bind to DNA along the adjacent
PRDIII and PRDI elements. Each of the four binding sites are separated
by a half turn of the DNA helix, thus enabling the opposite-facing
configuration of the two tandem molecules
.
Together,
the 32 basepair
sinusoidal region is called the PRD III-I element, and is composed of
nucleotides -96 to -64. In each PRD element, the 3’ end
contains a consensus sequence (AANNGAAA) and the 5’ end
contains a contains a nonconsensus site. IRF-3A and IRF-3C bind to noconsensus
sites
along
one side of the DNA
and IRF-3B and IRF-3D bind to consensus
sites
along
the opposite side
of the DNA. The DNA binding
domain of each IRF molecule is composed of three
helices, α1, α2 and α3
,
which
are adjacent to a 4-stranded
anti-parallel β-sheet
.
Three loops are
responsible for connecting these various elements. Loop L1 connects
β2
and α2, loop L2 connects α2 and α3, and
loop L3 connects β3 and β4.
III.
Specifics of DNA Binding
Each
IRF molecule binds to a region of DNA that
overlaps the region that
the adjacent IRF molecule binds to. IRF-A binds to PRD IV and PRD III,
IRF-B binds to PRD III, IRF-C binds to PRD III
and
PRD I
and
IRF-D binds to PRD I. IRF-3A and IRF-3B join head-to-tail and
associate with PRDIII as one complex. IRF-3C and IRF-3D bind together
in the same manner and link to PRDI in unison
The
curvatures of IRF-3A,
3B, 3C, and 3D are distinct from each other and allow for more
specificity in their associations with DNA. If the nonconsensus sites
are modified to become more similar to the consensus AANGAAA sequence,
IRF-3A and IRF-3B have a higher affinity for DNA. Genetic mutations,
such as the loss of a base, greatly reduces the transcription-inducing
capability of
the whole IRF-3 complex to a constitutive level. It is
critical to understand how damage to the DNA sequence (specifically the
consensus and nonconsensus regions) can affect the functionality of
IRF-3.
. The
two tandems form over
a two-nucleotide distance, while the association to form the full IRF-3
complex involves a binding over 3 nucleotides. By deleting one of the 3
bp separating Box B and Box C, enhancer activity is improved. The
variable binding network facilitates the flexibility of the protein-DNA
contacts.
α3,
the recognition helix
,
is positioned in the
major groove of DNA, parallel to the backbone. α3 makes
phosphate contacts
along the major groove. Arg78
and Arg86
are
important residues within this helix and allow for the flexibility
necessary for the molecule to bind to both consensus and nonconsensus
sequences of the enhancer
.
Loop
L1 is extremely flexible and
its positioning is highly based on the
presence or absence of DNA
.
IRF-3 binds to DNA in a manner such that loop L1 is positioned in the
minor groove. It contains His40,
which is interacts with DNA via water-mediated
hydrogen bonding
. Trp
38 is also located on
loop L1. Trp38
along with trp57,
which is on helix
α2, make phosphate contacts with the DNA backbone
. These
two residues, along
with three other tryptophans, are conserved across the DNA binding
domain of all the proteins in the IRF family. The Leu42
on loop L1
distinguishes IRF-3 from all other IRF proteins beause position 42 is
generally occupied by alanine
.
IV.
Effects on transcription
The
upregulation of the expression of the β-interferon as an
immunoresponse is initiated by IRF-3, the foundation of the
gene’s multi-protein enhaceosome complex. Under normal
circumstances, the 4 serine
residues
each subunit are phosphorylated, activating the protein.
.
All 4 molecules, IRF-3A, 3B, 3C, and 3D are equally necessary in the
enhancing activity of IRF-3. While binding sequence of the
tandems is relatively unimportant, the binding of only half of the
tandem is insufficient to produce the necessary β-interferon
response to a viral attack. To that extent, there is even some binding
cooperativity between the 4 elements of IRF-3
.
V.
References
Escalante, C. R. and Aggarwal,
A.
K. (2011). Structures of apo IRF-3 and IRF-7 DNA binding domains:
effect of loop L1 on DNA binding. Nucleic
Acids Research, 39(16),
7300-7307.
Escalante, C.R.,
Nistal-Villán
E., Shen, L., García-Sastre, A., Aggarwal, A. (2007).
Structure of IRF-3 bound to the PRDIII-I regulatory element of the
human interferon-β enhancer. Molecular
Cell. 26, 703-716.
Watson, J. D., Baker T. A., Bell,
S. P.,
Gann, A., Levine, M., and Losick, R. Molecular
Biology of the Gene. 6th ed.
Cold Spring Harbor: Cold Spring Harbor Laboratory, 2008. Print.
Back to Top