Imaging organelle membranes in live cells at the nanoscale with lipid-based fluorescent probes.

Understanding how organelles interact, exchange materials, assemble, disassemble, and evolve as a function of space, time, and environment is an exciting area at the very forefront of chemical and cell biology. Here, we bring attention to recent progress in the design and application of lipid-based tools to visualize and interrogate organelles in live cells, especially at super resolution. We highlight strategies that rely on modification of natural lipids or lipid-like small molecules ex cellula, where organelle specificity is provided by the structure of the chemically modified lipid, or in cellula using cellular machinery, where an enzyme labels the lipid in situ. We also describe recent improvements to the chemistry upon which lipid probes rely, many of which have already begun to broaden the scope of biological questions that can be addressed by imaging organelle membranes at the nanoscale.

Effects of nucleic acids and polyanions on dimer formation and DNA binding by bZIP and bHLHZip transcription factors.

A large fraction of known transcription factors form 2:1 complexes with DNA. In our studies of the assembly of such ternary (protein-protein-DNA) complexes formed by bZIP and bHLHZip proteins, we found that the proteins recognize DNA as monomers. Here we show that protein monomer-DNA complexes are favored at high DNA concentrations. Further, we show that, due to fast rates of association with protein monomers, DNA and other polyanions accelerate the rate of protein dimer formation. Finally, we find that DNA-assisted formation of protein dimers provides a mechanism by which dimeric transcription factors can rapidly discriminate between specific and nonspecific sites.

Electrostatic control of half-site spacing preferences by the cyclic AMP response element-binding protein CREB.

Basic region leucine zipper (bZIP) proteins represent a class of transcription factors that bind DNA using a simple, dimeric, alpha-helical recognition motif. The cAMP response element-binding protein (CREB) is a member of the CREB/ATF subfamily of bZIP proteins. CREB discriminates effectively in vivo and in vitro between the 10 bp cAMP response element (ATGACGTCAT, CRE) and the 9 bp activating protein 1 site (ATGACTCAT, AP-1). Here we describe an alanine scanning mutagenesis study designed to identify those residues within the CREB bZIP element that control CRE/AP-1 specificity. We find that the preference of CREB for the CRE site is controlled in a positive and negative way by acidic and basic residues in the basic, spacer and zipper segments. The CRE/AP-1 specificity of CREB is increased significantly by four glutamic acid residues located at positions 24, 28, 35 and 41; glutamic acid residues at positions 10 and 48 contribute in a more modest way. Specificity is decreased significantly by two basic residues located at positions 21 and 23; basic residues at positions 14, 18, 33 and 34 and V17 contribute in a more modest way. All of the residues that influence specificity significantly are located on the solvent-exposed face of the protein-DNA complex and likely participate in interactions between and among proteins, not between protein and DNA. The finding that the CRE/AP-1 specificity of CREB is dictated by the presence or absence of charged residues has interesting implications for how transcription factors seek and selectively bind sequences within genomic DNA.

Methodology for optimizing functional miniature proteins based on avian pancreatic polypeptide using phage display.

Synthetic genes for avian pancreatic polypeptide (aPP) and for the miniature DNA binding protein PPBR4 were cloned and expressed on the surface of M13 bacteriophage. We anticipate that these constructs will have utility optimizing the properties of miniature proteins based on aPP that result from our previously described protein grafting procedure.

Kinetic preference for oriented DNA binding by the yeast TATA-binding protein TBP.

In solution, the TATA box binding protein from S. cerevisiae (yTBP) is only minimally oriented when bound to the adenovirus major late promoter (AdMLP) and the yeast CYC1 promoter. At equilibrium, approximately 60% of the complexes are assembled in the orientation observed within crystal structures; 40% are assembled in the opposite orientation. Here we use stopped-flow fluorescence resonance energy transfer (FRET) to study the association kinetics of the two TBP.TATA box orientational isomers. Kinetics were determined by monitoring FRET between a unique tryptophan residue engineered into either the C- or the N-terminal stirrup of the conserved C-terminal subunit of yeast TBP (yTBPc) and an aminocoumarin moiety appended either upstream or downstream of the TATA box. Together, these constructs permitted a simultaneous yet independent monitor of the kinetics of TBP binding in both orientations. Not only did our results provide an independent confirmation of the free energy difference between the two orientational isomers, but they also showed that the orientational binding preference at equilibrium is a result of a faster association rate when TBP binds DNA in the orientation observed in the crystal structure.

Hepatitis B virus protein pX enhances the monomer assembly pathway of bZIP.DNA complexes.

The rapid and correct assembly of dimeric transcription factors on target DNA is essential for accurate transcriptional regulation. Here we ask how a viral accessory factor, hepatitis B virus X protein (pX), influences the rate and identity of the assembly pathway followed by members of the basic region leucine zipper (bZIP) transcription factor family. A combination of fluorescence polarization and fluorescence resonance energy transfer (FRET) experiments demonstrates unequivocally that pX does not increase the concentration of properly folded bZIP dimers in solution. Rather, fluorescence polarization and gel mobility shift experiments reveal that pX interacts directly with the basic-spacer segment of the bZIP peptide and stabilizes the complex composed of this monomer and target DNA. By stabilizing the intermediate formed along the monomer assembly pathway but not the one formed along the dimer pathway, pX enhances the equilibrium stability of a bZIP.DNA complex without changing the molecular mechanism used for complexation. Additional experiments reveal that pX decreases the kinetic specificity of certain bZIP proteins. To the extent that it is reflected at the transcriptional level, this loss in specificity could have far-reaching consequences for the host cell.

Hepatitis B virus X protein activates transcription by bypassing CREB phosphorylation, not by stabilizing bZIP-DNA complexes.

Although previous work has shown that the hepatitis B virus X protein (pX) stabilizes complexes between basic region leucine zipper (bZIP) proteins and target DNA, the relationship between enhanced binding and transcriptional activation has not been established. Here we show that interactions between CREB and pX, which coincidentally enhance DNA affinity, are necessary but not sufficient for increased transcriptional potency. Further, we show that transcriptional activation by pX requires a form of CREB in which Ser-133 is not phosphorylated. By stimulating the transcriptional potency of unphosphorylated CREB, pX can up-regulate the expression of cAMP-responsive genes implicated in hepatocyte proliferation, leading ultimately to the development of liver cancer after viral infection.

Kinetic studies of Fos.Jun.DNA complex formation: DNA binding prior to dimerization.

The bZIP proteins Fos and Jun bind DNA rapidly and with high affinity, forming a heteromeric complex that mediates activated transcription. Here we use stopped-flow fluorescence resonance energy transfer (FRET) to study the kinetic pathway by which Fos.Jun. DNA complexes assemble. Though dimerization of Fos and Jun occurs rapidly in the absence of DNA, the rate of dimerization is enhanced in the presence of DNA. Global analysis of the kinetic data shows that the favored DNA binding pathway is one is which the two protein monomers bind DNA sequentially and assemble their dimerization interface while bound to DNA.

Virtually unidirectional binding of TBP to the AdMLP TATA box within the quaternary complex with TFIIA and TFIIB.

BACKGROUND: The TATA box binding protein (TBP) is required by all three RNA polymerases for the promoter-specific initiation of transcription. All eukaryotic TBP-DNA complexes observed in crystal structures show the conserved C-terminal domain of TBP (TBPc) bound to the TATA box in a single orientation that is consistent with assembly of a preinitiation complex (PIC) possessing a unique polarity. The binding of TBP to the TATA box is believed to orient the PIC correctly on the promoter and can function as the rate-limiting step in PIC assembly. Previous work performed with TBP from Saccharomyces cerevisiae (yTBP) showed that, despite the oriented binding of eukaryotic TBP observed in crystal structures, yTBP in solution does not orient itself uniquely on the adenovirus major late promoter (AdMLP) TATA box. Instead, yTBP binds the AdMLP as a mixture of two orientational isomers that are related by a 180 degree rotation about the pseudo-dyad axis of the complex. In addition, these orientational isomers are not restricted to the 8 bp TATA box, but rather bind a distribution of sites that partially overlap the TATA box. Two members of the PIC, general transcription factor (TF) IIB and TFIIA individually enhance the orientational and axial specificity of yTBP binding to the TATA box, but fail to fix yTBP in a single orientation or a unique position on the promoter. RESULTS: We used an affinity cleavage assay to explore the combined effects of TFIIA and TFIIB on the axial and orientational specificity of yTBP. Our results show that the combination of TFIIA and TFIIB affixes yTBP in virtually a single orientation as well as a unique location on the AdMLP TATA box. Ninety-five percent of the quaternary TBP-TFIIA-TFIIB-TATA complex contained yTBP bound in the orientation expected on the basis of crystallographic and genetic experiments, and more than 70% is restricted axially to the 8 bp sequence TATAAAAG. CONCLUSIONS: Although yTBP itself binds to the TATA box without a high level of orientational or axial specificity, our data show that a small subset of general TFs are capable of uniquely orienting the PIC on the AdMLP. Our results, in combination with recent data concerning the pathway of PIC formation in yeast, suggest that transcription could be regulated during both early and late stages of PIC assembly by general factors (and the proteins to which they bind) that influence the position and orientation of TBP on the promoter.

Gene regulation: protein escorts to the transcription ball.

A new way by which the potency of a eukaryotic transcription factor can be regulated has been discovered, in which nuclear factors increase the concentration of the transcription factor's active form by modulating an otherwise unfavorable equilibrium between monomeric and dimeric forms of the protein.

DNA specificity enhanced by sequential binding of protein monomers.

Transcriptional activation often requires the rapid assembly of complexes between dimeric transcription factors and specific DNA sites. Here we show that members of the basic region leucine zipper and basic region helix-loop-helix zipper transcription factor families follow an assembly pathway in which two protein monomers bind DNA sequentially and form their dimerization interface while bound to DNA. Nonspecific protein or DNA competitors have little effect on the rate of assembly along this pathway, but slow a competing pathway in which preformed dimers bind DNA. The sequential monomer-binding pathway allows the protein to search for and locate a specific DNA site more quickly, resulting in greater specificity prior to equilibrium.

Preinitation complex assembly: potentially a bumpy path.

During 1996 and 1997, several chemical issues that arise in the early stages of preinitiation complex (PIC) formation were resolved. Kinetics experiments indicated that both TBP dimerization and DNA bending influence the rate of TBP-TATA box assembly. Affinity cleavage experiments indicated that TBP lacks the specificity to nucleate assembly of a properly oriented PIC. Finally, high-resolution structures provided the atomic detail of early intermediates in PIC formation.

Sequence determinants of the intrinsic bend in the cyclic AMP response element.

The cyclic AMP response element (CRE site, ATGACGTCAT) is the DNA target for transcription factors whose activities are regulated by cyclic AMP (1). Recently, we discovered that the CRE site is bent by 10-13 degrees toward the major groove (2). Little or no bend is detected in the related AP-1 site (ATGACTCAT), which differs from the CRE site by loss of a single, central, C.G base pair (2, 3). Here we describe experiments designed to identify which base pairs within the CRE site induce the bent structure in an attempt to understand the origins of the dramatically different conformations of the CRE and AP-1 sites. Our data indicate that the intrinsic CRE bend results from distortion within the TGA sequence found in each CRE half site (ATGAC). These two TGA sequences are located in phase with one another in the CRE sequence but are not (completely) in phase in the AP-1 sequence. This difference in phasing leads to the overall difference in bend as detected by gel (2) and cyclization methods (S. C. Hockings, J. D. Kahn, and D. M. Crothers, unpublished results; M. A. Fabian and A. Schepartz, unpublished results). Our results confirm earlier predictions of altered structure within TG steps, provide insight into the structural reorganizations induced in DNA by bZIP proteins, and lead to a revision of the relationship between the structures of the free and bZIP-bound forms of the CRE and AP-1 sites.

The role of a basic amino acid cluster in target site selection and non-specific binding of bZIP peptides to DNA.

The ability of a transcription factor to locate and bind its cognate DNA site in the presence of closely related sites and a vast array of non-specific DNA is crucial for cell survival. The CREB/ATF family of transcription factors is an important group of basic region leucine zipper (bZIP) proteins that display high affinity for the CRE site and low affinity for the closely related AP-1 site. Members of the CREB/ATF family share in common a cluster of basic amino acids at the N-terminus of their bZIP element. This basic cluster is necessary and sufficient to cause the CRE site to bend upon binding of a CREB/ATF protein. The possibility that DNA bending and CRE/AP-1 specificity were linked in CREB/ATF proteins was investigated using chimeric peptides derived from human CRE-BP1 (a member of the CREB/ATF family) and yeast GCN4, which lacks both a basic cluster and CRE/AP-1 specificity. Gain of function and loss of function experiments demonstrated that the basic cluster was not responsible for the CRE/AP-1 specificity displayed by all characterized CREB/ATF proteins. The basic cluster was, however, responsible for inducing very high affinity for non- specific DNA. It was further shown that basic cluster-containing peptides bind non-specific DNA in a random coil conformation. We postulate that the high non- specific DNA affinities of basic cluster-containing peptides result from cooperative electrostatic interactions with the phosphate backbone that do not require peptide organization.

Electrostatic mechanism for DNA bending by bZIP proteins.

Biology is replete with examples of protein-induced DNA bending, yet the forces responsible for bending have been neither established nor quantified. Mirzabekov and Rich proposed in 1979 that asymmetric neutralization of the anionic phosphodiester backbone by basic histone proteins could provide a thermodynamic driving force for DNA bending in the nucleosome core particle [Mirzabekov, A. D., & Rich, A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1118-1121]. Strauss and Maher lent support to this proposal in 1994 by demonstrating that replacement of six proximal phosphate residues with neutral methylphosphonates resulted in DNA bent spontaneously toward the neutralized face [Strauss, J. K., & Maher, L. J., III (1994) Science 266, 1829-1834; Strauss, J. K., Prakash, T. P., Roberts, C., Switzer, C., & Maher, L. J., III (1996) Chem. Biol. 3, 671-678; Strauss, J. K., Roberts, C.; Nelson, M. G.; Switzer, C., & Maher, J. L., III (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 9515-9520]. Here it is shown that bZIP proteins bend DNA via a mechanism involving direct contacts between one or two basic side chains and a symmetry-related pair of unique, nonbridging phosphate oxygens. The locations of these phosphates provide direct experimental support for a protein-induced bending mechanism based on asymmetric charge neutralization. This straightforward mechanism is compatible with many DNA-recognition motifs and may represent a general strategy for the assembly of protein-DNA complexes of defined stereochemistries.

Inhibition of Rev·RRE complexation by triplex tethered oligonucleotide probes.

We have described a class of molecules, called tethered oligonucleotide probes (TOPs), that bind RNA on the basis of both sequence and structure. TOPs consist of two short oligonucleotides joined by a tether whose length and composition may be varied using chemical synthesis. In a triplex TOP, one oligonucleotide recognizes a short single-stranded region in a target RNA through the formation of Watson-Crick base pairs; the other oligonucleotide recognizes a short double-stranded region through the formation of Hoogsteen base pairs. Binding of triplex TOPs to an HIV-1 Rev Response Element RNA variant (RREAU) was measured by competition electrophoretic mobility shift analysis. Triplex TOP.RREAU stabilities ranged between -9.6 and -6.1 kcal mol-1 under physiological conditions of pH, salt, and temperature. Although the most stable triplex TOP.RREAU complex contained 12 contiguous U.AU triple helical base pairs, complexes containing only six or nine triple helical base pairs also formed. Triplex TOPs inhibited formation of the RRE.Rev complex with IC50 values that paralleled the dissociation constants of the analogous triplex TOP.RREAU complexes. In contrast to results obtained with TOPs that target two single-stranded RRE regions, inhibition of Rev.RREAU complexation by triplex TOPs did not require pre-incubation of RREAU and a TOP: triplex TOPs competed efficiently with Rev for RREAU and inhibited RREAU.Rev complexation at equilibrium.