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Molecular Architecture of Transcription-Regulatory Protein
Complexes
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Tom K. W. Kerppola, Ph.D.
Associate Investigator,
University of Michigan Medical School
Biography...
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Summary:
Tom Kerppola is studying the molecular architecture of transcription factor
complexes and the structure, dynamics, and function of multiprotein
regulatory assemblies.
The selective regulation of gene expression in response to extracellular
signals requires a specific control mechanism for each gene. The differential
control of individual transcription units is achieved through combinatorial
interactions among multiple transcription-regulatory proteins at each
promoter. Interactions among transcription factors that bind to separate
regulatory elements require juxtaposition of complementary protein surfaces.
The arrangement of regulatory elements in the promoter region and the
organization of transcription factor complexes that bind to those elements
influence interactions among the transcription factors. Thus the unique
architecture of each promoter contributes to the exquisite specificity of
transcription regulation.
Many eukaryotic transcription-regulatory proteins form heterodimers with
related DNA-binding proteins. Such heterodimers frequently bind to regulatory
elements containing two symmetrical half-sites. They can potentially bind to
such regulatory elements in either of two opposite orientations. The protein
surfaces on opposite sides of heterodimers differ in their interactions with
other transcription factors. Thus the orientation of heterodimer binding may
influence transcription factor interactions and cooperative regulation of
transcriptional activity.
Fos-Jun Heterodimers Bind to Different Regulatory Elements in Opposite
Orientations
Heterodimers formed by the proto-oncogene transcription factors Fos and Jun
bind to AP-1 regulatory elements. The consensus AP-1 recognition sequence
consists of two symmetrical half-sites separated by an asymmetric central
base pair and flanked by nonsymmetrical sequences. To investigate the
orientation of heterodimer binding, we have developed a novel approach based
on fluorescence resonance energy transfer between a donor fluorophore linked
to either end of an oligonucleotide and an acceptor fluorophore linked to one
subunit of the heterodimer. Comparison of the efficiencies of energy transfer
from opposite ends of the oligonucleotide allows determination of the
half-site contacted by each subunit. The orientation preference is quantified
by calibration of the relative efficiencies of energy transfer, using
heterodimers with fixed differences in orientation preference. Examination of
the orientation of Fos-Jun heterodimer binding to different AP-1 sites
revealed that the heterodimer binds in opposite orientations to different
regulatory elements.
Sequences Flanking the AP-1 Site Influence the Orientation of
Heterodimer Binding
The effects of single–amino acid and single–base pair substitutions were
examined to identify the factors that determine the binding orientation.
Substitution of base pairs flanking the AP-1 recognition site reverses the
preferred orientation of Fos-Jun heterodimer binding. Fos and Jun do not
contact these base pairs in the x-ray crystal structure of the Fos–Jun–AP-1
complex. Substitution of charged amino acid residues outside the DNA contact
regions of Fos and Jun also affects the binding orientation. Thus indirect
recognition of flanking sequences by charged amino acid residues influences
the orientation of Fos-Jun heterodimer binding to the AP-1 site.
Fos and Jun bend DNA in opposite directions as a result of converse
electrostatic interactions with DNA flanking the AP-1 site. The orientation
of Fos-Jun heterodimer binding to different AP-1 sites correlates with
differences in DNA bending at those sites. Each subunit binds to the
half-site with a greater propensity to bend in the direction favored by that
protein.
To test the hypothesis that the opposite directions of DNA bending by Fos
and Jun affect the orientation of heterodimer binding, amino acid residues
that influence the direction of DNA bending were exchanged between Fos and
Jun. This exchange of DNA-bending properties reversed the orientation of
heterodimer binding. Heterodimers formed by proteins with similar bending
properties displayed little orientation preference, but heterodimers
containing subunits with different bending properties displayed an
orientation preference in proportion to the difference in DNA bending. Thus
the orientation of heterodimer binding is determined in part by matching the
opposite directions of DNA bending by Fos and Jun with differences in the
DNA-bending propensities of sequences on opposite sides of the AP-1 site.
The Orientation of Fos-Jun Heterodimer Binding and the Cooperativity
with NFAT1 Are Interrelated
The relationship between the orientation of heterodimer binding and
cooperative interactions with other transcription factors was investigated
for the Fos-Jun-NFAT1 (nuclear factor of activated T cells 1) complex. Fos
and Jun regulate cytokine gene expression in activated T cells in association
with NFAT family proteins. They bind cooperatively to composite regulatory
elements that contain degenerate AP-1 and NFAT recognition sequences. The
interaction with NFAT1 can reverse the orientation of Fos-Jun heterodimer
binding. This interaction also requires bending of DNA toward the interaction
interface. The exchange of three amino acid residues at the amino-terminal
ends of the leucine zippers of Fos and Jun is sufficient to reverse the
orientation of Fos-Jun heterodimer binding in the presence of NFAT1. These
residues therefore comprise the energetically dominant contact interface for
the asymmetric interaction between Fos-Jun and NFAT1.
The orientation of Fos-Jun heterodimer binding can also influence the
cooperativity of complex formation with NFAT1. Complexes formed by
heterodimers that bind in the same orientation in the presence and absence of
NFAT1 are more stable than complexes that bind in opposite orientations. Both
single–amino acid and single-nucleotide substitutions that affect the
orientation of heterodimer binding also alter the cooperativity of complex
formation. Compensatory substitutions in both protein and DNA restore both
the orientation of binding and cooperativity with NFAT1. Thus factors that
influence the orientation of heterodimer binding can also affect the
cooperativity of nucleoprotein complex formation.
The functional role of the orientation of heterodimer binding in mammalian
cells is being investigated. Inversion of heterodimer orientation may serve
as a switch to regulate transcriptional activity. This switch could be
controlled both by intrinsic and protein-induced variations in DNA structure,
by modifications that alter electrostatic interactions, or by contacts with transcription
factors. The overall architecture of the transcription-regulatory complex
represents the combined interactions among the individual transcription
factors and the promoter region. Changes in the conformations and
interactions of individual proteins may shift the equilibrium between active
and silent states.
Last updated: November 13, 2000
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