Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. Dimerization of BASIC LEUCINE ZIPPER (bZIP) TFs are critical for their functions, but the molecular mechanism underlying the DNA binding and functional specificity of homo-
Sequence‐specific binding by transcription factors (TFs) plays a significant role in the selection and regulation of target genes. At the protein:DNA interface, amino acid side‐chains construct a diverse physicochemical network of specific and non‐specific interactions, and seemingly subtle changes in amino acid identity at certain positions may dramatically impact TF:DNA binding. Variation of these specificity‐determining residues (SDRs) is a major mechanism of functional divergence between TFs with strong structural or sequence homology.
In this study, we employed a combination of high‐throughput specificity profiling by SELEX and Spec‐seq, structural modeling, and evolutionary analysis to probe the binding preferences of winged helix‐turn‐helix TFs belonging to the OmpR sub‐family in
We found that
Overall, our work provides evidence for a common structural “code” for sequence‐specific wHTH‐DNA interactions, and demonstrates that surprisingly modest residue changes can enable recognition of highly divergent sequence motifs. Further examination of SDR predictions will likely reveal additional mechanisms controlling the evolutionary divergence of this important class of transcriptional regulators.
- NSF-PAR ID:
- 10478004
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Quantitative Biology
- Volume:
- 6
- Issue:
- 1
- ISSN:
- 2095-4689
- Format(s):
- Medium: X Size: p. 68-84
- Size(s):
- ["p. 68-84"]
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract versus heterodimers remains elusive. To address this gap, we present the double DNA Affinity Purification-sequencing (dDAP-seq) technique that maps heterodimer binding sites on endogenous genomic DNA. Using dDAP-seq we profile twenty pairs of C/S1 bZIP heterodimers and S1 homodimers inArabidopsis and show that heterodimerization significantly expands the DNA binding preferences of these TFs. Analysis of dDAP-seq binding sites reveals the function of bZIP9 in abscisic acid response and the role of bZIP53 heterodimer-specific binding in seed maturation. The C/S1 heterodimers show distinct preferences for the ACGT elements recognized by plant bZIPs and motifs resembling the yeast GCN4cis -elements. This study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation. -
more » « less
Abstract Tumor necrosis factor receptor‐associated factors (TRAFs) constitute a family of adapter proteins that act in numerous signaling pathways important in human biology and disease. The MATH domain of TRAF proteins binds peptides found in the cytoplasmic domains of signaling receptors, thereby connecting extracellular signals to downstream effectors. Beyond several very general motifs, the peptide binding preferences of TRAFs have not been extensively characterized, and differences between the binding preferences of TRAF paralogs are poorly understood. Here we report a screening system that we established to explore TRAF peptide‐binding specificity using deep mutational scanning of TRAF‐peptide ligands. We displayed single‐ and double‐mutant peptide libraries based on the TRAF‐binding sites of CD40 or TANK on the surface of
Escherichia coli and screened them for binding to TRAF2, TRAF3, and TRAF5. Enrichment analysis of the library sequencing results showed differences in the permitted substitution patterns in the TANK versus CD40 backgrounds. The three TRAF proteins also demonstrated different preferences for binding to members of the CD40 library, and three peptides from that library that were analyzed individually showed striking differences in affinity for the three TRAFs. These results illustrate a previously unappreciated level of binding specificity between these close paralogs and demonstrate that established motifs are overly simplistic. The results from this work begin to outline differences between TRAF family members, and the experimental approach established herein will enable future efforts to investigate and redesign TRAF peptide‐binding specificity. -
more » « less
Abstract The regulation of floral organ identity was investigated using a forward genetic approach in five floral homeotic mutants of
Thalictrum , a noncore eudicot. We hypothesized that these mutants carry defects in the floral patterning genes. Mutant characterization comprised comparative floral morphology and organ identity gene expression at early and late developmental stages, followed by sequence analysis of coding and intronic regions to identify transcription factor binding sites and protein–protein interaction (PPI) motifs. Mutants exhibited altered expression of floral MADS‐box genes, which further informed the function of paralogs arising from gene duplications not found in reference model systems. The ensuing modified BCE models for the mutants supported instances of neofunctionalization (e.g., B‐class genes expressed ectopically in sepals), partial redundancy (E‐class), or subfunctionalization (C‐class) of paralogs. A lack of deleterious mutations in the coding regions of candidate floral MADS‐box genes suggested thatcis ‐regulatory ortrans ‐acting mutations are at play. Consistent with this hypothesis, double‐flower mutants had transposon insertions or showed signs of transposon activity in the regulatory intron ofAGAMOUS (AG ) orthologs. Single amino acid substitutions were also found, yet they did not fall on any of the identified DNA binding or PPI motifs. In conclusion, we present evidence suggesting that transposon activity and regulatory mutations in floral homeotic genes likely underlie the striking phenotypes of theseThalictrum floral homeotic mutants. -
more » « less
Abstract We report herein a study on the impact of bifacial peptide nucleic acid (bPNA) amino acid composition and backbone modification on DNA binding. A series of bPNA backbone variants with identical net charge were synthesized to display either 4 or 6 melamine (M) bases. These bases form thymine‐melamine‐thymine (TMT) base‐triples, resulting in triplex hybrid stem structures with T‐rich DNAs. Analyses of 6 M bPNA‐DNA hybrids suggested that hybrid stability was linked to amino acid secondary structure propensities, prompting a more detailed study in shorter 4 M bPNAs. We synthesized 4 M bPNAs predisposed to adopt helical secondary structure via helix‐turn nucleation in 7‐residue bPNAs using double‐click covalent stapling. Generally, hybrid stability improved upon stapling, but amino acid composition had a more significant effect. We also pursued an alternative strategy for bPNA structural preorganization by incorporation of residues with strong backbone amide conformational preferences such as 4
R ‐ and 4S ‐fluoroprolines. Notably, these derivatives exhibited an additional improvement in hybrid stability beyond both unsubstituted proline bPNA analogues and the helically patterned bPNAs. Overall, these findings demonstrate the tunability of bPNA‐DNA hybrid stability through bPNA backbone structural propensities and amino acid composition. -
Geminiviruses possess single-stranded, circular DNA genomes and control the transcription of their late genes, including BV1 of many bipartite begomoviruses, through transcriptional activation by the early expressing AC2 protein. DNA binding by AC2 is not sequence-specific; hence, the specificity of AC2 activation is thought to be conferred by plant transcription factors (TFs) recruited by AC2 in infected cells. However, the exact TFs AC2 recruits are not known for most viruses. Here, we report a systematic examination of the BV1 promoter (PBV1) of the mungbean yellow mosaic virus (MYMV) for conserved promoter motifs. We found that MYMV PBV1 contains three abscisic acid (ABA)-responsive elements (ABREs) within its first 70 nucleotides. Deleting these ABREs, or mutating them all via site-directed mutagenesis, abolished the capacity of PBV1 to respond to AC2-mediated transcriptional activation. Furthermore, ABRE and other related ABA-responsive elements were prevalent in more than a dozen Old World begomoviruses we inspected. Together, these findings suggest that ABA-responsive TFs may be recruited by AC2 to BV1 promoters of these viruses to confer specificity to AC2 activation. These observations are expected to guide the search for the actual TF(s), furthering our understanding of the mechanisms of AC2 action.more » « less
