Degenerate Binding Site Research Articles

Insights into the functions and RNA binding of Trypanosoma brucei ZC3H22, RBP9 and DRBD7.

Trypanosoma brucei is unusually reliant on mRNA-binding proteins to control mRNA fate, because its protein-coding genes lack individual promoters. We here focus on three trypanosome RNA-binding proteins. ZC3H22 is specific to Tsetse fly forms, RBP9 is preferentially expressed in bloodstream forms; and DRBD7 is constitutively expressed. Depletion of RBP9 or DRBD7 did not affect bloodstream-form trypanosome growth. ZC3H22 depletion from procyclic forms caused cell clumping, decreased expression of genes required for cell growth and proliferation, and increased expression of some epimastigote markers. Apart from decreases in mRNAs encoding enzymes of glucose metabolism, levels of most ZC3H22-bound transcripts were unaffected by ZC3H22 depletion. We compared ZC3H22, RBP9 and DRBD7 RNA binding with that of 16 other RNA-binding proteins. ZC3H22, PUF3 and ERBP1 show a preference for ribosomal protein mRNAs. RBP9 preferentially binds mRNAs that are more abundant in bloodstream forms than in procyclic forms. RBP9, ZC3H5, ZC3H30 and DRBD7 prefer mRNAs with long coding regions; UBP1-associated mRNAs have long 3'-untranslated regions; and RRM1 prefers mRNAs with long 3'or 5'-untranslated regions. We suggest that proteins that prefer long mRNAs may have relatively short or degenerate binding sites, and that preferences for A or U increase binding in untranslated regions.

KIRMES: kernel-based identification of regulatory modules in euchromatic sequences

Motivation: Understanding transcriptional regulation is one of the main challenges in computational biology. An important problem is the identification of transcription factor (TF) binding sites in promoter regions of potential TF target genes. It is typically approached by position weight matrix-based motif identification algorithms using Gibbs sampling, or heuristics to extend seed oligos. Such algorithms succeed in identifying single, relatively well-conserved binding sites, but tend to fail when it comes to the identification of combinations of several degenerate binding sites, as those often found in cis-regulatory modules. Results: We propose a new algorithm that combines the benefits of existing motif finding with the ones of support vector machines (SVMs) to find degenerate motifs in order to improve the modeling of regulatory modules. In experiments on microarray data from Arabidopsis thaliana, we were able to show that the newly developed strategy significantly improves the recognition of TF targets. Availability: The python source code (open source-licensed under GPL), the data for the experiments and a Galaxy-based web service are available at http://www.fml.mpg.de/raetsch/suppl/kirmes/ Contact: ed.gpm.negnibeut@ibes Supplementary information: Supplementary data are available at Bioinformatics online.

KIRMES: kernel-based identification of regulatory modules in euchromatic sequences

Motivation: Understanding transcriptional regulation is one of the main challenges in computational biology. An important problem is the identification of transcription factor (TF) binding sites in promoter regions of potential TF target genes. It is typically approached by position weight matrix-based motif identification algorithms using Gibbs sampling, or heuristics to extend seed oligos. Such algorithms succeed in identifying single, relatively well-conserved binding sites, but tend to fail when it comes to the identification of combinations of several degenerate binding sites, as those often found in cis-regulatory modules.Results: We propose a new algorithm that combines the benefits of existing motif finding with the ones of support vector machines (SVMs) to find degenerate motifs in order to improve the modeling of regulatory modules. In experiments on microarray data from Arabidopsis thaliana, we were able to show that the newly developed strategy significantly improves the recognition of TF targets.Availability: The python source code (open source-licensed under GPL), the data for the experiments and a Galaxy-based web service are available at http://www.fml.mpg.de/raetsch/suppl/kirmes/Contact: sebi@tuebingen.mpg.deSupplementary information: Supplementary data are available at Bioinformatics online.

Structures of Human Pumilio with Noncognate RNAs Reveal Molecular Mechanisms for Binding Promiscuity

Pumilio is a founder member of the evolutionarily conserved Puf family of RNA-binding proteins that control a number of physiological processes in eukaryotes. A structure of human Pumilio (hPum) Puf domain bound to a Drosophila regulatory sequence showed that each Puf repeat recognizes a single nucleotide. Puf domains in general bind promiscuously to a large set of degenerate sequences, but the structural basis for this promiscuity has been unclear. Here, we describe the structures of hPum Puf domain complexed to two noncognate RNAs, CycB(reverse) and Puf5. In each complex, one of the nucleotides is ejected from the binding surface, in effect, acting as a "spacer." The complexes also reveal the plasticity of several Puf repeats, which recognize noncanonical nucleotides. Together, these complexes provide a molecular basis for recognition of degenerate binding sites, which significantly increases the number of mRNAs targeted for regulation by Puf proteins in vivo.

Entanglement of static and flying qubits in degenerate mesoscopic systems

The entanglement of flying qubits with static qubits in the solid state is important in the development of quantum computing. It has recently been shown theoretically that the spin-dependent scattering of a propagating electron from a bound electron is sufficient to give full entanglement between the qubits embodied in their respective spins [J. H. Jefferson et al., Europhys. Lett. 74, 764 (2006); D. Gunlycke et al., J. Phys.: Condens. Matter 18, S851 (2006)]. In this paper a generalized, real-space Anderson model is introduced for a quasi-one-dimensional structure consisting of a binding site coupled to ideal leads. Degeneracy of both the binding site and the leads is incorporated to represent, for example, conduction band degeneracy in carbon nanotubes. The model is used to calculate the spin-dependent scattering behavior and resultant static-flying qubit entanglement created by the scattering process. Degeneracy (and more generally, multiplicity) in the binding site gives rise to inelastic scattering processes. In the elastic scattering regime, a degenerate binding site gives rise to antiresonant structures in the transmission spectrum. Additional degeneracy in the leads restricts this effect to the second set of leads, raising the possibility of spin filtration, though this is eliminated in the inelastic scattering regime. Degeneracy in the binding site also gives rise to multiple resonances in transmission that will improve the probability of obtaining entangled pairs relative to the nondegenerate case. This effect is maximized when the components of the degenerate binding site are symmetrically coupled to the leads.

Identification of Genomic Sites That Bind the Drosophila Suppressor of Hairy-wing Insulator Protein

Eukaryotic genomes are divided into independent transcriptional domains by DNA elements known as insulators. The gypsy insulator, a 350-bp element isolated from the Drosophila gypsy retrovirus, contains twelve degenerate binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Su(Hw) associates with over 500 non-gypsy genomic sites, the functions of which are largely unknown. Using a bioinformatics approach, we identified 37 putative Su(Hw) insulators (pSIs) that represent regions containing clustered matches to the gypsy insulator Su(Hw) consensus binding sequence. The majority of these pSIs contain fewer than four Su(Hw) binding sites, with only seven showing in vivo Su(Hw) association, as demonstrated by chromatin immunoprecipitation. To understand the properties of the pSIs, these elements were tested for enhancer-blocking capabilities using a transgene assay system. In a complementary set of experiments, effects of the pSIs on transcriptional regulation of genes at the natural genomic location were determined. Our data suggest that pSIs have complex genomic functions and, in some cases, establish insulators. These studies provide the first direct evidence that the Su(Hw) protein contributes to the regulation of gene expression in the Drosophila genome through the establishment of endogenous insulators.

Scanning mutagenesis using T4 DNA ligase and short degenerate DNA oligonucleotides containing tri-nucleotide mismatches.

Scanning mutagenesis is an attractive tool for protein structure-function correlation analysis. With one round of this method it is possible to obtain a library containing all possible single-residue mutants of the protein of interest. The practical application of this approach is currently limited by the large number and cost of the required 30-35mer oligonucleotides. As an alternative, we studied the ligation of shorter DNA oligonucleotides (6-11mer) containing a degenerate binding site and a desired mutation mismatch to a nested set of megaprimers annealed to the gene of interest. T4 DNA ligase was able to perform this task, and the obtained ligation products were elongated by DNA polymerase. The effectiveness of ligation depends on the length of the random binding site of the mutagenic oligonucleotide, on its molar excess over the template-primer complex and on the position of the mismatching tri-nucleotide insert with respect to the joining site. The secondary structure of the DNA template close to the joining site also influences the ligation yield. Mismatching oligonucleotides, protected by a 3'-phosphate group, were joined to a nested set of megaprimers, the latter being obtained by a novel procedure called reversible chain termination, i.e., termination of the dsDNA synthesis with ddNTP followed by the subsequent removal of the incorporated ddNMP with exonuclease III. T7 sequenase 2.0 DNA polymerase elongated the ligation products after the 3'-phosphate protection group was removed with T4 polynucleotide kinase, resulting in the incorporation of a specific tri-nucleotide mismatch into dsDNA. This sequence of reactions serves as the basis for a novel scanning mutagenesis procedure.

Enhancer Blocking by the Drosophila gypsy Insulator Depends Upon Insulator Anatomy and Enhancer Strength

Insulators are specialized DNA sequences that prevent enhancer-activated transcription only when interposed between an enhancer and its target promoter. The Drosophila gypsy retrotransposon contains an insulator composed of 12 degenerate binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein that are separated by AT-rich DNA possessing sequence motifs common to matrix/scaffold attachment regions (MARs/SARs). To further understand mechanisms of insulator function, the parameters required for the gypsy insulator to prevent enhancer-activated transcription were examined. Synthetic binding regions were created by reiteration of a single Su(Hw) binding site that lacked the MAR/SAR motifs. These synthetic binding regions reconstituted insulator activity, suggesting that the property of enhancer blocking may be distinct from matrix association. We found that the number and spacing of Su(Hw) binding sites within the gypsy insulator, as well as the strength of the enhancer to be blocked, were important determinants of insulator function. These results provide a link between transcription and insulation, suggesting that these processes may be mechanistically interconnected.