Tetraplex structure of fission yeast telomeric DNA and unfolding of the tetraplex on the interaction with telomeric DNA binding protein Pot1

To understand the regulation mechanism of fission yeast telomeric DNA, we analysed the structural properties of Gn: d(GnTTAC) (n=2-6) and 4Gn: d(GnTTAC)4 (n=3 and 4), and their interaction with the single-stranded telomeric DNA binding domain of telomere-binding protein Pot1 (Pot1DBD). G4, G5 and G6 formed a parallel tetraplex in contrast with no tetraplex formation by G2 and G3. Also, 4G4 adopted only an antiparallel tetraplex in spite of a mixture of parallel and antiparallel tetraplexes of 4G3. The variety of tetraplex structures was governed by the number of consecutive guanines in a single copy and the number of repeats. The antiparallel tetraplex of 4G4 became unfolded upon the interaction with Pot1DBD. The interaction with mutant Pot1DBD proteins revealed that the ability to unfold the antiparallel tetraplex was strongly correlated with the specific binding affinity for the single-stranded telomeric DNA. The result suggests that the decrease in the free single strand upon the complex formation with Pot1DBD may shift the equilibrium from the tetraplex to the single strand, which may cause the tetraplex unfolding. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex is required for telomerase-mediated telomere regulation.     

Drosophila cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis

Translational control is a critical process in the spatio-temporal restriction of protein production. In Drosophila oogenesis, translational repression of oskar (osk) RNA during its localization to the posterior pole of the oocyte is essential for embryonic patterning and germ cell formation. This repression is mediated by the osk 3' UTR binding protein Bruno (Bru), but the underlying mechanism has remained elusive. Here, we report that an ovarian protein, Cup, is required to repress precocious osk translation. Cup binds the 5'-cap binding translation initiation factor eIF4E through a sequence conserved among eIF4E binding proteins. A mutant Cup protein lacking this sequence fails to repress osk translation in vivo. Furthermore, Cup interacts with Bru in a yeast two-hybrid assay, and the Cup-eIF4E complex associates with Bru in an RNA-independent manner. These results suggest that translational repression of osk RNA is achieved through a 5'/3' interaction mediated by an eIF4E-Cup-Bru complex.     

Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine.

Using CD spectroscopy, guanine tetraplex formation was studied with short DNA fragments in which cytosine residues were systematically added to runs of guanine either at the 5' or 3' ends. Potassium cations induced the G-tetraplex more easily with fragments having the guanine run at the 5' end, which is just an opposite tendency to what was reported for (G+T) oligonucleotides. However, the present (G+C) fragments simultaneously adopted other conformers that complicated the analysis. We demonstrate that repeated freezing/thawing, performed at low ionic strength, is a suitable method to exclusively stabilize the tetraplex in the (G+C) DNA fragments. In contrast to KCl, the repeated freeze/thaw cycles better stabilized the tetraplex with fragments having the guanine run on the 3' end. The tendency of guanine blocks to generate the tetraplex destabilized the d(G5).d(C5) duplex whose strands dissociated, giving rise to a stable tetraplex of (dG5) and single-stranded (dC5). In contrast to d(G3C3) and d(G5C5), repeated freezing/thawing induced the tetraplex even with the self-complementary d(C3G3) or d(C5G5); hence the latter oligonucleotides preferred the tetraplex to the apparently very stable duplex. The tetraplexes only included guanine blocks while the 5' end cytosines interfered neither with the tetraplex formation nor the tetraplex structure.     

Evidence for allosteric coupling between the ribosome and repressor binding sites of a translationally regulated mRNA

Escherichia coli ribosomal protein S4 is a translational repressor regulating the expression of four ribosomal genes in the alpha operon. In vitro studies have shown that the protein specifically recognizes an unusual mRNA pseudoknot secondary structure which links sequences upstream and downstream of the ribosome binding site for rpsM (S13) [Tang, C. K., & Draper, D. E. (1989) Cell 57, 531]. We have prepared fusions of the rpsM translational initiation site and lacZ that allows us to detect repression in cells in which overproduction of S4 repressor can be induced. Twenty-five mRNA sequence variants have been introduced into the S13-lacZ fusions and the levels of translational repression measured. Sets of compensating base changes confirm the importance of the pseudoknot secondary structure for translational repression. An A residue in a looped, single-stranded sequence is also required for S4 recognition and may contact S4 directly. Comparison of translational repression levels and S4 binding constants for the set of mRNA mutations show that nine mutants are repressed much more weakly than predicted from their affinity for S4; in extreme cases no repression can be detected for variants with unchanged S4 binding. We suggest that the mRNA contains functionally distinct ribosome and repressor binding sites that are allosterically coupled. Mutations can relieve translational repression by disrupting the linkage between the two sites without altering S4 binding. This proposal assigns to the mRNA a more active role in mediating translational repression than found in other translational repression systems.     

Fission yeast telomeric DNA binding protein Pot1 has the ability to unfold tetraplex structure of telomeric DNA

To understand the regulation mechanism of fission yeast telomeric DNA, we analyzed the structural properties of 4Gn: d(G(n)TTAC)(4) (n = 3, 4) and their interaction with the single-stranded telomeric DNA binding domain of telomere-binding protein Pot1 (Pot1DBD). 4G4 adopted only an antiparallel tetraplex in spite of a mixture of parallel and antiparallel tetraplexes of 4G3. The antiparallel tetraplex of 4G4 became unfolded upon the interaction with Pot1DBD. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex is required for telomerase-mediated telomere regulation.     

Nucleotide-dependent binding of the gene 4 protein of bacteriophage T7 to single-stranded DNA

The gene 4 protein of bacteriophage T7 is a multifunctional enzyme that catalyzes (i) the hydrolysis of nucleoside 5'-triphosphates, (ii) the synthesis of tetraribonucleotide primers at specific recognition sequences on a DNA template, and (iii) the unwinding of duplex DNA. All three activities depend on binding of gene 4 protein to single-stranded DNA followed by unidirectional 5' to 3' translocation of the protein (Tabor, S., and Richardson, C. C. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 205-209). Binding of gene 4 protein to single-stranded DNA, assayed by retention of DNA-protein complexes on nitrocellulose filters, is random with regard to DNA sequence. Although gene 4 protein does not bind to duplex DNAs, the presence of a 240-nucleotide-long single-stranded tail on a 7200-base pair duplex DNA molecule is sufficient for gene 4 protein to cause retention of the DNA on a filter. The binding reaction requires, in addition to MgCl2, the presence of a nucleoside 5'-triphosphate, but binding is not dependent on hydrolysis; nucleoside 5'-diphosphate will substitute for nucleoside 5'-triphosphate. Of the eight common nucleoside triphosphates, dTTP promotes optimal binding. The half-life of the gene 4 protein-DNA complex depends on both the secondary structure of the DNA and on whether or not the nucleoside 5'-triphosphate cofactor can be hydrolyzed. Using the nonhydrolyzable nucleoside 5'-triphosphate analog, beta,gamma-methylene dTTP, the half-life of the gene 4 protein-DNA complex is greater than 80 min. In the presence of the hydrolyzable nucleoside 5'-triphosphate, dTTP, the half-life of the gene 4 protein-DNA complex using circular M13 DNA is at least 4 times longer than that observed using linear M13 DNA.     

Oxytricha telomeric nucleoprotein complexes reconstituted with synthetic DNA.

The telomere binding protein from macronuclei of Oxytricha nova binds macronuclear DNA in vitro, protecting the 3'-terminal single-stranded (T4G4)2 tail from chemical and enzymatic probes. We have used synthetic oligodeoxynucleotides to study the binding properties of the telomere protein. It binds at the 3' end of single-stranded oligonucleotides that have the sequence (T4G4)n, where n greater than or equal to 2, reconstituting the methylation protection seen with macronuclear DNA. Three oligonucleotide.protein complexes are resolved in nondenaturing gels, all specific for this sequence. Single-stranded oligonucleotides that have one or more repeats of the sequence C4A4 are also recognized, forming a single complex. The dissociation constant for (T4G4)4 is about 19 nM, and for macronuclear DNA is at least 20-fold lower. The basis for this difference is not fully understood, but it is not simply due to the absence of a (C4A4)2.5.(G4T4)2.5 region on the oligonucleotide. Transversions of T's to A's or of G's to C's in the 3' tail portion prevent binding. Changing T's to dU's does not prevent binding, indicating that the hydrophobic 5-methyl group is not required for binding as had been suggested from the salt-stability of the complex. The properties of the DNA-protein complex suggest a revised model for telomere synthesis in Oxytricha.     

In vitro binding of the bacteriophage f1 gene V protein to the gene II RNA-operator and its DNA analog.

We have investigated the binding of the f1 single-stranded DNA-binding protein (gene V protein) to DNA oligonucleotides and RNA synthesized in vitro. The first 16 nucleotides of the f1 gene II mRNA leader sequence were previously identified as the gene II RNA-operator; the target to which the gene V protein binds to repress gene II translation. Using a gel retardation assay, we find that the preferential binding of gene V protein to an RNA carrying the gene II RNA-operator sequence is affected by mutations which abolish gene II translational repression in vivo. In vitro, gene V protein also binds preferentially to a DNA oligonucleotide whose sequence is the DNA analog of the wild-type gene II RNA-operator. Therefore, the gene V protein recognizes the gene II mRNA operator sequence when present in either an RNA or DNA context.     

Autoregulation of gene expression: Quantitative evaluation of the expression and function of the bacteriophage T4 gene 32 (single-stranded DNA binding) protein system

The free concentration of bacteriophage T4-coded gene 32 (single-stranded DNA binding) protein in the cell is autoregulated at the translational level during T4 infection of Escherichia coli. The control of the synthesis of this protein reflects the following progression of net (co-operative) binding affinities for the various potential nucleic acid binding targets present: single-stranded DNA > gene 32 mRNA > other T4 mRNAs ? double-stranded DNA. In this paper we show that the free concentration of gene 32 protein is maintained at 2 to 3 μm, and use the measured binding parameters for gene 32 protein, extrapolated to intracellular conditions, to provide a quantitative molecular interpretation of this system of control of gene expression. These results are then further utilized to define the specific autoregulatory binding sequence (translational operator site) on the gene 32 mRNA as a uniquely unstructured finite binding lattice terminated by elements of secondary structure not subject to melting by gene 32 protein at the autoregulated concentration, and to predict how this site must differ from those found on other T4 messenger RNAs. It is shown that these predictions are fully consistent with available T4 DNA sequence data. The control of free protein concentration as a method of genome regulation is discussed in terms of other systems to which these approaches may apply.     

Tetraplex formation of surface-immobilized human telomere sequence probed by surface plasmon resonance using single-stranded DNA binding protein

Many sequences in genomic DNA are able to form unique tetraplex structures. Such structures are involved in a variety of important cellular processes and are emerging as a new class of therapeutic targets for cancers and other diseases. Screening for molecules targeting the tetraplex structure has been explored using such sequences immobilized on solid surfaces. Immobilized nucleic acids, in certain situations, may better resemble the molecules under in vivo conditions. In this report, we studied the formation of tetraplex structure of both the G-rich and C-rich strands of surface-immobilized human telomere sequence by surface plasmon resonance using the single-stranded DNA binding protein from Escherichia coli as probe. We demonstrate how the formation of G-quadruplex and i-motif could be probed under various conditions by this sequence-universal method. Our results also show that immobilization destabilized the tetraplex structure.     

Human Ku antigen tightly binds and stabilizes a tetrahelical form of the Fragile X syndrome d(CGG)n expanded sequence

Hairpin and tetrahelical structures of a d(CGG)(n) sequence in the FMR1 gene have been implicated in its expansion in fragile X syndrome. The identification of tetraplex d(CGG)(n) destabilizing proteins (Fry, M., and Loeb, L. A.(1999) J. Biol. Chem. 274, 12797-12803; Weisman-Shomer, P., Naot, Y., and Fry, M. (2000) J. Biol. Chem. 275, 2231-2238) suggested that proteins might modulate d(CGG)(n) folding and aggregation. We assayed human TK-6 lymphoblastoid cell extracts for d(CGG)(8) oligomer binding proteins. The principal binding protein was identified as Ku antigen by its partial amino acid sequence and antigenicity. The purified 88/75-kDa heterodimeric Ku bound with similar affinities (K(d) approximately 1. 8-10.2 x 10(-9) mol/liter) to double-stranded d(CGG)(8).d(CCG)(8), hairpin d(CGG)(8), single-stranded d(CII)(8), or tetraplex structures of telomeric or IgG switch region sequences. However, Ku associated more tightly with bimolecular G'2 tetraplex d(CGG)(8) (K(d) approximately 0.35 x 10(-9) mol/liter). Binding to Ku protected G'2 d(CGG)(8) against nuclease digestion and impeded its unwinding by the tetraplex destabilizing protein qTBP42. Stabilization of d(CGG)(n) tetraplex domains in FMR1 by Ku or other proteins might promote d(CGG) expansion and FMR1 silencing.     

The structure of a ribosomal protein S8/spc operon mRNA complex

In bacteria, translation of all the ribosomal protein cistrons in the spc operon mRNA is repressed by the binding of the product of one of them, S8, to an internal sequence at the 5' end of the L5 cistron. The way in which the first two genes of the spc operon are regulated, retroregulation, is mechanistically distinct from translational repression by S8 of the genes from L5 onward. A 2.8 A resolution crystal structure has been obtained of Escherichia coli S8 bound to this site. Despite sequence differences, the structure of this complex is almost identical to that of the S8/helix 21 complex seen in the small ribosomal subunit, consistent with the hypothesis that autogenous regulation of ribosomal protein synthesis results from conformational similarities between mRNAs and rRNAs. S8 binding must repress the translation of its own mRNA by inhibiting the formation of a ribosomal initiation complex at the start of the L5 cistron.     

Identification of proteins associating with poly(A)-binding-protein mRNA.

Synthesis of poly(A)-binding protein is regulated at the translational level. We have investigated the binding of proteins to this mRNA on the premise that the protein(s) of the mRNP complex may be involved in regulating the expression of the mRNA. We found the first 243 nucleotides of the 5' untranslated region to contain sequences essential for RNP formation. A large, single-stranded bulge structure encompassing stretches rich in adenine nucleotides and a potential stem-loop domain appear to be the primary sites for protein binding. Removal of the 243-nucleotide segment results in a drastic reduction in protein binding and a concomitant increase in translational efficiency in vitro. We suggest that proteins binding to this region, including poly(A)-binding protein itself, may be essential for regulating translation of this mRNA.     

eIF4E-binding protein regulation of mRNAs with differential 5'-UTR secondary structure: a polyelectrostatic model for a component of protein-mRNA interactions

Control of translation in eukaryotes is complex, depending on the binding of various factors to mRNAs. Available data for subsets of mRNAs that are translationally up- and down-regulated in yeast eIF4E-binding protein (4E-BP) deletion mutants are coupled with reported mRNA secondary structure measurements to investigate whether 5'-UTR secondary structure varies between the subsets. Genes with up-regulated translational efficiencies in the caf20Δ mutant have relatively high averaged 5'-UTR secondary structure. There is no apparent wide-scale correlation of RNA-binding protein preferences with the increased 5'-UTR secondary structure, leading us to speculate that the secondary structure itself may play a role in differential partitioning of mRNAs between eIF4E/4E-BP repression and eIF4E/eIF4G translation initiation. Both Caf20p and Eap1p contain stretches of positive charge in regions of predicted disorder. Such regions are also present in eIF4G and have been reported to associate with mRNA binding. The pattern of these segments, around the canonical eIF4E-binding motif, varies between each 4E-BP and eIF4G. Analysis of gene ontology shows that yeast proteins containing predicted disordered segments, with positive charge runs, are enriched for nucleic acid binding. We propose that the 4E-BPs act, in part, as differential, flexible, polyelectrostatic scaffolds for mRNAs.     

How bacterial ribosomal protein L20 assembles with 23 S ribosomal RNA and its own messenger RNA

In bacteria, the expression of ribosomal proteins is often feedback-regulated at the translational level by the binding of the protein to its own mRNA. This is the case for L20, which binds to two distinct sites of its mRNA that both resemble its binding site on 23 S rRNA. In the present work, we report an NMR analysis of the interaction between the C-terminal domain of L20 (L20C) and both its rRNA- and mRNA-binding sites. Changes in the NMR chemical shifts of the L20C backbone nuclei were used to show that the same set of residues are modified upon addition of either the rRNA or the mRNA fragments, suggesting a mimicry at the atomic level. In addition, small angle x-ray scattering experiments, performed with the rRNA fragment, demonstrated the formation of a complex made of two RNAs and two L20C molecules. A low resolution model of this complex was then calculated using (i) the rRNA/L20C structure in the 50 S context and (ii) NMR and small angle x-ray scattering results. The formation of this complex is interesting in the context of gene regulation because it suggests that translational repression could be performed by a complex of two proteins, each interacting with the two distinct L20-binding sites within the operator.     

Requirements for primer synthesis by bacteriophage T7 63-kDa gene 4 protein. Roles of template sequence and T7 56-kDa gene 4 protein.

Gene 4 of bacteriophage T7 encodes two proteins, a 63-kDa protein and a colinear 56-kDa protein, that are essential for synthesis of leading and lagging strands during DNA replication. The gene 4 proteins together catalyze the synthesis of oligoribonucleotides, pppACC(C/A) or pppACAC, at the single-stranded DNA sequences 3'-CTGG(G/T)-5' or 3'-CTGTG-5', respectively. Purified 56-kDa protein has helicase activity, but no primase activity. In order to study 63-kDa gene 4 protein free of 56-kDa gene 4 protein, mutations were introduced into the internal ribosome-binding site responsible for the translation of the 56-kDa protein. The 63-kDa gene 4 protein was purified 16,000-fold from Escherichia coli cells harboring an expression vector containing the mutated gene 4. Purified 63-kDa gene 4 protein has primase, helicase, and single-stranded DNA-dependent dTTPase activities. The constraints of primase recognition sequences, nucleotide substrate requirements, and the effects of additional proteins on oligoribonucleotide synthesis by the 63-kDa gene 4 protein have been examined using templates of defined sequence. A three-base sequence, 3'-CTG-5', is necessary and sufficient to support the synthesis of pppAC dimers. dTTP hydrolysis is essential for oligoribonucleotide synthesis. Addition of a 7-fold molar excess of 56-kDa gene 4 protein to 63-kDa protein increases the number of oligoribonucleotides synthesized by 63-kDa protein 100-fold. The increase in oligonucleotides results predominantly from an increase in the synthesis of tetramers, with relatively little change in the synthesis of dimers and trimers. The presence of 56-kDa protein also causes 63-kDa protein to synthesize "pseudo-templated" pppACCCC pentamers at the recognition sequence 3'-CTGGG-5'. T7 gene 2.5 protein, a single-stranded DNA binding protein, increases the total number of oligoribonucleotides synthesized by 63-kDa gene 4 protein on single-stranded M13 DNA, but has no effect on the ratio of dimers to trimers and tetramers.     

DNA recognition and binding by the Euplotes telomere protein.

The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule.