Telomeres of polytene chromosomes in a ciliated protozoan terminate in duplex DNA loops

The end of a telomeric DNA sequence isolated from a polytene chromosome of a hypotrichous ciliate folds back and hybridizes with downstream telomeric sequence to form a t loop that is stable in the absence of protein and DNA cross-linking. The single-stranded, telomeric DNA sequence at the end of a macronuclear molecule does not form a t loop but, instead, is complexed with a heterodimeric, telomere-binding protein. Thus, two mechanisms for capping the ends of DNA molecules are used in the same cell.

Structure and function in rhodopsin: Topology of the C-terminal polypeptide chain in relation to the cytoplasmic loops*

Cysteine mutagenesis and site-directed spin labeling in the C-terminal region of rhodopsin have been used to probe the local structure and proximity of that region to the cytoplasmic loops. Each of the native amino acids in the sequence T335–T340 was replaced with Cys, one at a time. The sulfhydryl groups of all mutants reacted rapidly with the sulfhydryl reagent 4,4′-dithiodipyridine, which indicated a high degree of solvent accessibility. Furthermore, to probe the proximity relationships, a series of double Cys mutants was constructed. One Cys in all sets was at position 338 and the other was at a position in the sequence S240–V250 in the EF interhelical loop, at position 65 in the AB interhelical loop, or at position 140 in the CD interhelical loop. In the dark state, no significant disulfide formation was observed between C338 and C65 or C140 under the conditions used, whereas a relatively rapid disulfide formation was observed between C338 and C242 or C245. Spin labels in the double Cys mutants showed the strongest magnetic interactions between the nitroxides attached to C338 and C245 or C246. Light activation of the double mutant T242C/S338C resulted in slower disulfide formation, whereas interactions between nitroxides at C338 and C245 or C246 decreased. These results suggest the proximity of the C-terminal residue C338 to residues located on the outer face of a cytoplasmic helical extension of the F helix with an apparent increase of distance upon photoactivation.

A Polymer Model for the Structural Organization of Chromatin Loops and Minibands in Interphase Chromosomes

A quantitative model of interphase chromosome higher-order structure is presented based on the isochore model of the genome and results obtained in the field of copolymer research. G1 chromosomes are approximated in the model as multiblock copolymers of the 30-nm chromatin fiber, which alternately contain two types of 0.5- to 1-Mbp blocks (R and G minibands) differing in GC content and DNA-bound proteins. A G1 chromosome forms a single-chain string of loop clusters (micelles), with each loop ∼1–2 Mbp in size. The number of ∼20 loops per micelle was estimated from the dependence of geometrical versus genomic distances between two points on a G1 chromosome. The greater degree of chromatin extension in R versus G minibands and a difference in the replication time for these minibands (early S phase for R versus late S phase for G) are explained in this model as a result of the location of R minibands at micelle cores and G minibands at loop apices. The estimated number of micelles per nucleus is close to the observed number of replication clusters at the onset of S phase. A relationship between chromosomal and nuclear sizes for several types of higher eukaryotic cells (insects, plants, and mammals) is well described through the micelle structure of interphase chromosomes. For yeast cells, this relationship is described by a linear coil configuration of chromosomes.

A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops

Colicin D has long been thought to stop protein synthesis in infected Escherichia coli cells by inactivating ribosomes, just like colicin E3. Here, we show that colicin D specifically cleaves tRNAsArg including four isoaccepting molecules both in vivo and in vitro. The cleavage occurs in vitro between positions 38 and 39 in an anticodon loop with a 2′,3′-cyclic phosphate end, and is inhibited by a specific immunity protein. Consistent with the cleavage of tRNAsArg, the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine. Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity in vivo together with the tRNAArg-cleaving activity both in vivo and in vitro. These findings show that colicin D directly cleaves cytoplasmic tRNAsArg, which leads to impairment of protein synthesis and cell death. Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp. Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.

Real-time quantitative measurement of autocrine ligand binding indicates that autocrine loops are spatially localized

Autocrine ligands are important regulators of many normal tissues and have been implicated in a number of disease states, including cancer. However, because by definition autocrine ligands are synthesized, secreted, and bound to cell receptors within an intrinsically self-contained “loop,” standard pharmacological approaches cannot be used to investigate relationships between ligand/receptor binding and consequent cellular responses. We demonstrate here a new approach for measurement of autocrine ligand binding to cells, using a microphysiometer assay originally developed for investigating cell responses to exogenous ligands. This technique permits quantitative measurements of autocrine responses on the time scale of receptor binding and internalization, thus allowing investigation of the role of receptor trafficking and dynamics in cellular responses. We used this technique to investigate autocrine signaling through the epidermal growth factor receptor by transforming growth factor alpha (TGFα) and found that anti-receptor antibodies are far more effective than anti-ligand antibodies in inhibiting autocrine signaling. This result indicates that autocrine-based signals can operate in a spatially restricted, local manner and thus provide cells with information on their local microenvironment.

Interlocked feedback loops contribute to the robustness of the Neurospora circadian clock

Interlocked feedback loops may represent a common feature among the regulatory systems controlling circadian rhythms. The Neurospora circadian feedback loops involve white collar-1 (wc-1), wc-2, and frequency (frq) genes. We show that WC-1 and WC-2 proteins activate the transcription of frq gene, whereas FRQ protein plays dual roles: repressing its own transcription, probably by interacting with the WC-1/WC-2 complex, and activating the expression of both WC proteins. Thus, they form two interlocked feedback loops: one negative and one positive. We establish the physiological significance of the interlocked positive feedback loops by showing that the levels of WC-1 and WC-2 determine the robustness and stability of the clock. Our data demonstrate that with WC-1 being the limiting factor in the WC-1/WC-2 complex, the greater the levels of WC-1 and WC-2, the higher the level of the FRQ oscillation and the more robust the overt rhythms. Our data also show that, despite considerable changes in the levels of WC-1, WC-2, and FRQ, the period of the clock has been limited to a small range, suggesting that the interlocked circadian feedback loops are also important for determining the circadian period length of the clock.

Interplay of structure and disorder in cochaperonin mobile loops.

Protein-protein interactions typically are characterized by highly specific interfaces that mediate binding with precisely tuned affinities. Binding of the Escherichia coli cochaperonin GroES to chaperonin GroEL is mediated, at least in part, by a mobile polypeptide loop in GroES that becomes immobilized in the GroEL/GroES/nucleotide complex. The bacteriophage T4 cochaperonin Gp31 possesses a similar highly flexible polypeptide loop in a region of the protein that shows low, but significant, amino acid similarity with GroES and other cochaperonins. When bound to GroEL, a synthetic peptide representing the mobile loop of either GroES or Gp31 adopts a characteristic bulged hairpin conformation as determined by transferred nuclear Overhauser effects in NMR spectra. Thermodynamic considerations suggest that flexible disorder in the cochaperonin mobile loops moderates their affinity for GroEL to facilitate cycles of chaperonin-mediated protein folding.

Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.

Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides.

The role of surface loops (residues 204-216 and 627-646) in the motor function of the myosin head.

A characteristic feature of all myosins is the presence of two sequences which despite considerable variations in length and composition can be aligned with loops 1 (residues 204-216) and 2 (residues 627-646) in the chicken myosin-head heavy chain sequence. Recently, an intriguing hypothesis has been put forth suggesting that diverse performances of myosin motors are achieved through variations in the sequences of loops 1 and 2 [Spudich, J. (1994) Nature (London) 372, 515-518]. Here, we report on the study of the effects of tryptic digestion of these loops on the motor and enzymatic functions of myosin. Tryptic digestions of myosin, which produced heavy meromyosin (HMM) with different percentages of molecules cleaved at both loop 1 and loop 2, resulted in the consistent decrease in the sliding velocity of actin filaments over HMM in the in vitro motility assays, did not affect the Vmax, and increased the Km values for actin-activated ATPase of HMM. Selective cleavage of loop 2 on HMM decreased its affinity for actin but did not change the sliding velocity of actin in the in vitro motility assays. The cleavage of loop 1 and HMM decreased the mean sliding velocity of actin in such assays by almost 50% but did not alter its affinity for HMM. To test for a possible kinetic determinant of the change in motility, 1-N6-ethenoadenosine diphosphate (epsilon-ADP) release from cleaved and uncleaved myosin subfragment 1 (S1) was examined. Tryptic digestion of loop 1 slightly accelerated the release of epsilon-ADP from S1 but did not affect the rate of epsilon-ADP release from acto-S1 complex. Overall, the results of this work support the hypothesis that loop 1 can modulate the motor function of myosin and suggest that such modulation involves a mechanism other than regulation of ADP release from myosin.

DNA loops induced by cooperative binding of transcriptional activator proteins and preinitiation complexes.

DNA conformational changes are essential for the assembly of multiprotein complexes that contact several DNA sequence elements. An approach based on atomic force microscopy was chosen to visualize specific protein-DNA interactions occurring on eukaryotic class II nuclear gene promoters. Here we report that binding of the transcription regulatory protein Jun to linearized plasmid DNA containing the consensus AP-1 binding site upstream of a class II gene promoter leads to bending of the DNA template. This binding of Jun was found to be essential for the formation of preinitiation complexes (PICs). The cooperative binding of Jun and PIC led to looping of DNA at the protein binding sites. These loops were not seen in the absence of either PICs, Jun, or the AP-1 binding site, suggesting a direct interaction between DNA-bound Jun homodimers and proteins bound to the core promoter. This direct visualization of functional transcriptional complexes confirms the theoretical predictions for the mode of gene regulation by trans-activating proteins.