User:Irfan Saleh/sandbox 1
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Article Name1. Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9). 2. Structural Insight into the Mechanism of Double-Stranded RNA Processing by Ribonuclease III. Protein NameName: 2EZ6 Description (2EZ6) = A. aeolicus ribonuclease III(RNaseIII) Number of Amino Acids in (2EZ6) = 218 Number of Nucleic Acids in (2EZ6) = 56 Catalytic Valley Length and Width = 50Å, 20Å The Type of 2EZ6 PDB = dsRNA RNA Description of (2EZ6) = Double-Stranded RNA (dsRNA) The Crystal structure of A. aeolicus RNaseIII-dsRBD, which forms a complex with dsRNA has the 2EZ6 PDBID code. Furthermore, information regarding the complex is published in the article listed under the article name and in Figure 6 of the original article 1 [3] The crystal structure of A. aeolicus RNaseIII-dsRBD was first discovered in the Aquifex aeolicus. Aquifex aeolicus is a bacterium that has similar shape to a rod and it is 2 to 6 micrometers long in length The 2EZ6 protein has a sequence shape and containing a 4 chain structure. The binding mode of A. aeolicus RNaseIII-dsRBD with short RNA duplex was originally observed in the crystal structure of A. aeolicus RNaseIII-dsRBD. The A. aeolicus structure shows that the bound short RNA duplex is surrounded by two dsRBD domains on one side and two RNaseIII domains on the other side.
Background InformationAs mentioned earlier that 2EZ6 is the crystal structure for the protein RNaseIII. However, the complete structure of RNaseIII is consisted of the double stranded RNA binding domain that binds to RNA. RNaseIII has an active important role in the RNA silencing inducing complex. More specifically, the protein 2EZ6 is a model of the siRNA duplex and it is surrounded by two dsRBDs in the front and in the back without any stereo clashes it is covered by D1 and D2 of RHA helicase core. The model itself represents a functioning model for recognizing the siRNA duplex. The model also displays the partial unwinding of the siRNA by the RHA protein. The RNA helicase is the loading factor for the RISC complex. RHA has dsRBD1/dsRBD2 domains, which facilitates in binding to siRNA. Furthermore, such binding mode was observed in the crystal structure of A. aeolicus RNaseIII-dsRBD in complex with short RNA duplex. The RHA protein helps in facilitating the RISC assembly, which enables the binding of siRNA duplex and its interactions with Ago2, TRBP and Dicer. However, the structural and functional features of RHA in dsRNA binding and RISC assembly are largely unknown. Therefore the original study investigated and determined structural insights of siRNA duplex recognition and the RISC assembly that is facilitated by RHA dsRBD domains. As a critical factor RISC plays important functions in RNAi pathways. Study showed that human RNA helicase A also known as DHX9 functions as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains. RISC represses the translation output and specific sequences of mRNA in a sequence-specific manner. The two double stranded RNA binding domains (dsRBD1/dsRBD2) are required for RISC association and such association is mediated by dsRNA. The crystal structure analysis further revealed that the RHA protein cooperatively works with dsRBDS to recognized siRNAs. This evidence suggested that RHA promotes RNA silencing by promoting the formation of active RISC. The study discoveries also revealed that that two dsRBD domains are an absolute requirement for interaction with RISC while the helicase core is not absolutely needed to facilitate the formation of active RISC in humans.
Protein FunctionMembers of the ribonuclease III also known as RNase III family are double-stranded RNA (dsRNA) and as family it is highly conserved. Some endoribonucleases are characterized in their active centers by a signature motif. The products of RNase III have a two-base 3' overhang. The primary functions of RNase III are consisted of RNA processing and post-transcriptional gene-expression control[4]. RNase III has helped in understanding the importance of the role of the Dicer in RNA interference. Dicer produces small interfering RNAs. The RNA interference is a range of class of gene-silencing systems initiated by dsRNA. Based on increasing molecular weight and complexity of the polypeptide chain the RNase III family can be divided into four classes. The four classes are bacterial RNase III, Saccharomyces cerevisiae Rnt1p, Drosophila melanogaster Drosha, and Homo sapiens Dicer. The bacterial RNase III proteins, such as Escherichia coli RNase III (Ec-RNase III) and Aquifex aeolicus RNase III (Aa-RNase III) are composed of an endonuclease domain (endoND) followed by a dsRNA binding domain (dsRBD).The homodimeric Ec-RNase III was first discovered in 1968 and ever since it has become the most extensively studied member of the family. RNase III has the ability to affect the gene expression in either of two ways: as a processing enzyme or as a binding protein. As a processing enzyme, RNase III cleaves both natural and synthetic dsRNA into small duplex products averaging 10–18 base pairs in length. As a binding protein, RNase III binds and stabilizes certain RNAs including the suppression of certain genes. The RNase III-product complex has a 7 residue linker within the protein, which facilitates induced fit in protein-RNA recognition. A pattern of protein-RNA interactions known as four RNA binding motifs in RNase III and three protein-interacting boxes in dsRNA, are responsible for substrate specificity. Meanwhile conserved amino acid residues and divalent cations are responsible for scissile-bond cleavage. Studying the structure of RNase III is important, because it can be applied to other structures of the RNase III family. Here the crystal structure of an RNase III-product complex is displayed in scenes.
RelevanceThe major importance of the PDB 2EZ6 is that it is part of the RISC assembly [5], which codes for RNA silencing. RNA silencing refers to a conserved sequence-specific gene regulation mechanism initiated by small RNA molecules. RNA silencing has critical roles in many important biological processes in eukaryotes, including host gene regulation and defense against invading foreign nucleic acids. More specifically the application of this protein is integral in the small RNA processing and small RNA-mediated gene regulation. Both RNA processing and gene regulation are integral factors of gene therapy, preventing viral and cancerous mutations in the human body. Furthermore, researchers examined the change in gene expression in relationship to-disease to predict its effect on the disease based on early changes in gene expression after irradiation[6]. Therefore, gene silencing and the regulation of certain genes could be an easy solution to the prevention of deadliest diseases. (eQTL) Definition: Expression quantitative trait loci (eQTLs) are genomic loci that contribute to variation in expression levels of mRNAs. In another study eQTL mapping was applied to identify associations between gene dysregulation and single nucleotide polymorphism (SNP) genotypes in glioblastoma multiform (GBM). A set of 532,954 SNPs were evaluated as predictors of the expression levels of 22,279 expression probes. The final number of significant probes was reduced to 9257 after identifying SNPs associated with fold change in expression level rather than raw expression levels in the tumor and making some adjustments for confounding variables in the experiment. Essentially, the expression probes were involved in signal transduction, transcription regulation, membrane function, and cell cycle regulation. These methods and results indicated that there are many fixed position on the loci that can serve as key part of regulatory pathways associated with GBM. [7].
Structural HighlightsThe is the complete crystal structure of the(RNase III) This is the scene for the dsRBD1 binding to RNA This is the scene for the side chain and including all residues The scene is the list of 8 Residues makes up large part of the(RNase III) active site This scene shows the RNaseIII The binding of dsRBD to RNA through H-bonding References
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