Yang-Gyun Kim

Solution Structure of a Luteoviral P1-P2 Frameshifting mRNA Pseudoknot

A hairpin-type messenger RNA pseudoknot from pea enation mosaic virus RNA1 (PEMV-1) regulates the efficiency of programmed -1 ribosomal frameshifting. The solution structure and 15N relaxation rates reveal that the PEMV-1 pseudoknot is a compact-folded structure composed almost entirely of RNA triple helix. A three nucleotide reverse turn in loop 1 positions a protonated cytidine, C(10), in the correct orientation to form an A((n-1)).C(+).G-C(n) major groove base quadruple, like that found in the beet western yellows virus pseudoknot and the hepatitis delta virus ribozyme, despite distinct structural contexts. A novel loop 2-loop 1 A.U Hoogsteen base-pair stacks on the C(10)(+).G(28) base-pair of the A(12).C(10)(+).G(28)-C(13) quadruple and forms a wedge between the pseudoknot stems stabilizing a bent and over-rotated global conformation. Substitution of key nucleotides that stabilize the unique conformation of the PEMV-1 pseudoknot greatly reduces ribosomal frameshifting efficacy.

Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

Zα is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zα is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1) , a candidate enzyme for nuclear pre-mRNA editing in mammals. Zα is conserved in ADAR1 from many species; in each case, there is a second similar motif,Zβ, separated from Zα by a more divergent linker. To investigate the structure-function relationship ofZα, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zα is part of a domain, Zab, of 229 amino acids (residues 133–361 in human ADAR1). This domain contains both Zα and Zβas well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133–209), containing only Zα, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zβ, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zαand Zβ act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

Mutational study reveals that tertiary interactions are conserved in ribosomal frameshifting pseudoknots of two luteoviruses.

Expression of the putative replicase of potato leafroll virus (PLRV) is regulated by -1 ribosomal frameshifting in which a primary viral transcript has two overlapping open reading frames (ORFs). A region of 39 nt at the junction of the two ORFs is essential for frameshifting to occur. It has been shown to harbor two signals, one active on the level of the primary structure, termed the slippery sequence, and one component that forms a secondary or tertiary level structure, described as either a pseudoknot or a stem-loop motif. We have performed extensive site-directed mutagenesis of the frameshifting region and analyzed individual mutants for their ability to promote -1 frameshifting in vitro. Detailed comparison of our results with analogous mutants in the frameshifting region of the evolutionarily related beet western yellow virus, for which a crystal structure is available, unequivocally argues for the pseudoknot to be the structural motif necessary for the frameshifting function in PLRV transcripts. Mutations in PLRV that affect putative pseudoknot-specific tertiary-base interactions drastically affect frameshifting activity. In addition, a specific deletion mutant was identified that displayed PLRV wild-type frameshifting activity with only 22 nt available for pseudoknot formation.

The structures of non-CG-repeat Z-DNAs co-crystallized with the Z-DNA-binding domain, hZαADAR1

The Z-DNA conformation preferentially occurs at alternating purine-pyrimidine repeats, and is specifically recognized by Zα domains identified in several Z-DNA-binding proteins. The binding of Zα to foreign or chromosomal DNA in various sequence contexts is known to influence various biological functions, including the DNA-mediated innate immune response and transcriptional modulation of gene expression. For these reasons, understanding its binding mode and the conformational diversity of Zα bound Z-DNAs is of considerable importance. However, structural studies of Zα bound Z-DNA have been mostly limited to standard CG-repeat DNAs. Here, we have solved the crystal structures of three representative non-CG repeat DNAs, d(CACGTG)2, d(CGTACG)2 and d(CGGCCG)2 complexed to hZαADAR1 and compared those structures with that of hZαADAR1/d(CGCGCG)2 and the Zα-free Z-DNAs. hZαADAR1 bound to each of the three Z-DNAs showed a well conserved binding mode with very limited structural deviation irrespective of the DNA sequence, although varying numbers of residues were in contact with Z-DNA. Z-DNAs display less structural alterations in the Zα-bound state than in their free form, thereby suggesting that conformational diversities of Z-DNAs are restrained by the binding pocket of Zα. These data suggest that Z-DNAs are recognized by Zα through common conformational features regardless of the sequence and structural alterations.

The interaction between Z-DNA and the Zab domain of double-stranded RNA adenosine deaminase characterized using fusion nucleases.

Zab is a structurally defined protein domain that binds specifically to DNA in the Z conformation. It consists of amino acids 133–368 from the N terminus of human double-stranded RNA adenosine deaminase, which is implicated in RNA editing. Zab contains two motifs with related sequence, Zα and Zβ. Zα alone is capable of binding Z-DNA with high affinity, whereas Zβ alone has little DNA binding activity. Instead, Zβ modulates Zα binding, resulting in increased sequence specificity for alternating (dCdG) n as compared with (dCdA/dTdG) n . This relative specificity has previously been demonstrated with short oligonucleotides. Here we demonstrate that Zab can also bind tightly to (dCdG) n stabilized in the Z form in supercoiled plasmids. Binding was assayed by monitoring cleavage of the plasmids using fusion nucleases, in which Z-DNA-binding peptides from the N terminus of double-stranded RNA adenosine deaminase are linked to the nuclease domain of FokI. A fusion nuclease containing Zα shows less sequence specificity, as well as less conformation specificity, than one containing Zab. Further, a construct in which Zβ has been replaced in Zab with Zα, cleaves Z-DNA regions in supercoiled plasmids more efficiently than the wild type but with little sequence specificity. We conclude that in the Zab domain, both Zα and Zβ contact DNA. Zα contributes contacts that produce conformation specificity but not sequence specificity. In contrast, Zβ contributes weakly to binding affinity but discriminates between sequences of Z-DNAs.

Base extrusion is found at helical junctions between right- and left-handed forms of DNA and RNA

Base extrusion is a major structural feature at the junction between B- and Z-DNA (the B–Z junction) where a base pair is broken, and the two bases are extruded from the double helix. Despite the demonstration of base extrusion at the B–Z junction, it is not clear whether a similar base extrusion occurs at other types of junctions involving the left-handed Z conformation. Here, we investigate structural changes of bases at three Z-form junctions: DNA B–Z and Z–Z and RNA A–Z junctions. By monitoring fluorescently labeled duplex nucleic acids using 2-aminopurines at various positions relative to the junction point, we show that base extrusion occurs not only at the DNA B–Z junction, but also at the RNA A–Z and DNA Z–Z junctions. Our data suggest that base extrusion is a general feature of Z-form nucleic-acid junctions.

Sequence, genomic organization and functional expression of the murine tRNA-specific adenosine deaminase ADAT1

We have recently identified the first mammalian tRNA-specific adenosine deaminase human ADAT1, a member of the ADAR family of RNA editing enzymes. This protein is responsible for the first step of the unique A(37) to m(1)I(37) modification in eukaryotic tRNA(Ala). Here, we present the genomic structure of murine ADAT1 and the functional expression of mADAT1 cDNA. In mouse, as well as in human, ADAT1 is expressed from a single copy gene. The coding region of the mADAT1 gene is spread over nine exons, covering approximately 30kb of genomic DNA and encodes a protein of 499 amino acids. Overall, mADAT1 shares 81% nucleotide homology and 87.5% protein homology with the human ortholog. The recombinant mouse protein is active specifically and with a high efficiency on human tRNA(Ala) in vitro. Its genomic organization is compared to the structures of the sequence-related, pre-mRNA specific adenosine deaminases ADAR1 and ADAR2.

Characterization of DNA-binding activity of Zα domains from poxviruses and the importance of the β-wing regions in converting B-DNA to Z-DNA

The E3L gene is essential for pathogenesis in vaccinia virus. The E3L gene product consists of an N-terminal Zα domain and a C-terminal double-stranded RNA (dsRNA) binding domain; the left-handed Z-DNA-binding activity of the Zα domain of E3L is required for viral pathogenicity in mice. E3L is highly conserved among poxviruses, including the smallpox virus, and it is likely that the orthologous Zα domains play similar roles. To better understand the biological function of E3L proteins, we have investigated the Z-DNA-binding behavior of five representative Zα domains from poxviruses. Using surface plasmon resonance (SPR), we have demonstrated that these viral Zα domains bind Z-DNA tightly. Ability of ZαE3L converting B-DNA to Z-DNA was measured by circular dichroism (CD). The extents to which these Zαs can stabilize Z-DNA vary considerably. Mutational studies demonstrate that residues in the loop of the β-wing play an important role in this stabilization. Notably the Zα domain of vaccinia E3L acquires ability to convert B-DNA to Z-DNA by mutating amino acid residues in this region. Differences in the host cells of the various poxviruses may require different abilities to stabilize Z-DNA; this may be reflected in the observed differences in behavior in these Zα proteins.

Genomic clustering of tRNA-specific adenosine deaminase ADAT1 and two tRNA synthetases.

Abstract. Human tRNA-specific adenosine deaminase (hADAT1) specifically converts A37 in the anticodon loop of human tRNAAla to inosine via a hydrolytic deamination mechanism. The enzyme is related to a family of RNA editing enzymes (ADARs) specific for pre-mRNA, and it has been cloned based on its sequence homology to the catalytic domain of ADARs. In the present study we have analyzed the 5′-flanking sequence of the murine ADAT1 gene, revealing that the first transcribed exon is located 1.1 kb downstream from the polyadenylation site of lysyl tRNA synthetase (KARS). The close proximity is conserved in the human genome with an intergenic distance of 5.5 kb. We determined the complete cDNA sequence as well as exon/intron organization of murine KARS. Significant sequence similarities between KARS and ADAT1 are apparent within their substrate interaction domains. Radiation hybrid panel analysis mapped human ADAT1 and human KARS to region q22.2–22.3 of Chromosome (Chr) 16 with alanyl tRNA synthetase (AARS) positioned centromeric to the KARS and ADAT1 genes. 16q22–24 has recently been recognized as a susceptibility candidate locus for several autoimmune inflammatory diseases. The clustering of three tRNA specific genes, of which two are specific for tRNAAla, may indicate their evolutionary relatedness or common factors involved in regulating their expression.