Sandbox Reserved 931
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| - | + | ==LINE-1 ORF1 Protein== | |
| + | Long interspersed nuclear element 1 (LINE1) is a non-long terminal repeat (non-LTR) type retrotransposon in mammals that engages in retrotransposition - a process which randomly inserts LINE1's own coding sequence into the host genome, increasing the genome size and causing genomic instability. The LINE1 gene encodes the open reading frame 1 protein (L1ORF1p), which localizes to large L1 ribonucleoprotein particles, stress granules and nucleus (ref), and is required and necessary for retrotransposition. | ||
| - | ==Stabilization of the Trimeric Structure== | + | ==Introduction== |
| + | After transcription of the LINE1 gene, the same RNA transcript is both translated and is the template for reverse transcription. This RNA transcript contains two open reading frames: ORF1 and ORF2, which codes for two proteins, ORF1p and ORF2p. L1ORF1p contains the RNA packing and delivery functions, and L1ORF2p contains the enzymatic machinery for reverse transcription. Translation of both of these open reading frames is necessary for retrotransposition. The reverse transcribed LINE1 is integrated into the host genome in a different place than the original gene. | ||
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| + | L1ORF1p localizes to ribonucleoprotein particles, stress granules and nucleus. Although the protein has general affinity to nucleic acids, it displays a strong <i>cis</i> preference, which makes it bind the primary encoding RNA transcript<ref>[http://mcb.asm.org/content/21/4/1429.short Wei, Gilber, Ooi, Lawler, Ostertag, Kazazian, Boeke and Moran]</ref>. The role of L1ORF1p in retrotransposition is to protect the transcript from degradation and to help transport it to the nucleus. | ||
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| + | ==Structure== | ||
| + | <StructureSection load='2YKO' size='500' side='right' caption='3D structure view' scene=''> | ||
| + | The majority of the L1ORF1 structure was solved by X-ray crystallography<ref>[http://www.nature.com/nsmb/journal/v18/n9/abs/nsmb.2097.html Khazina, Truffault, Büttner, Schmidt, Coles and Weichenrieder]</ref>. The crystallized part of the protein is a 338-residue, 40 kDa chain composed of <scene name='57/579701/Orf1p_overall_structure/2'>3 domains</scene>: long N-terminal alpha-helical domain, central RNA-recognition and binding domain (RRM) and a C-terminal domain (CTD). The N-terminal residues were not crystallized, but based on previous atomic force microscopy experiments<ref>[http://www.sciencedirect.com/science/article/pii/S0022283605016396 Basame, Li, Howard, Branciforte, Keller and Martin]</ref> they are expected to extend the alpha-helix by approximately 50 residues. The domains are connected with two linker regions responsible for structure flexibility. The overall structure of the protein forms an L-shaped pocket formed by N terminal helix and central region with the flexible C-terminal domain “capping” the binding pocket (link to monomer). | ||
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| + | ===Stabilization of the Trimeric Structure=== | ||
| + | L1ORF1p forms <scene name='57/579701/Orf1p_overall_structure/3'>homotrimers</scene> with unusually long alpha helices (residues 111-153), which create a coiled-coil structure. The very long N-terminal helices are stabilized by three structural features: | ||
| + | *Coordination of <scene name='57/579701/Orf1p_overall_structure/6'>two chloride anions</scene> on the hydrophobic interface inside the coiled-coil structure by three asparagines (Asn142) and three arginines (Arg135). These promote the trimeric state of the coiled-coil domain; | ||
| + | *Externally stabilizing <scene name='57/579701/Orf1p_overall_structure/7'>hydrogen bonds</scene> between coiled-coil helices along the hydrophilic face of the helices; | ||
| + | *Water exclusion interactions within the internal hydrophobic face of the helices in the coiled-coil domain. | ||
| + | The trimerization is additionally stabilized on the C-terminal side of the molecule by 1 hydrogen bond between <scene name='57/579701/Orf1p_overall_structure/11'>each RRM</scene> domain, while the CTD regions remain more flexible and do not interact with one another. This might play a significant role in the accommodation of RNA molecules or during retrotransciption. | ||
| + | |||
| + | ===Nucleic Acid Binding Surfaces=== | ||
| + | L1ORF1p is an RNA-interacting molecule, although its structure in the RNA-bound state was not crystallized. Careful mapping of protein surface electrostatic potential indicated the likely sites of RNA accommodation. The protein contains two major grooves with positively-charged surface whose affinity to negatively-charged RNA is highest: | ||
| + | *Horizontal cleft between the <scene name='57/579701/Orf1p_overall_structure/9'>RRM and CTD domains</scene> of each monomer, where the local electrostatic potential reaches up to +15 kT/e; | ||
| + | *Vertical, wide groove between monomers, with somewhat lower positive local electrostatics. | ||
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| + | It has been demonstrated by mutagenesis that arginine residues 220, 235 and 261<ref>[http://nar.oxfordjournals.org/content/39/13/5611.full Evans, Peddigari, Chaurasiya, Williams and Martin]</ref> that are on the surface between the RRM and CTD domains are indispensable for the RNA affinity of the trimer. Therefore, in the RNA-bound state, the RNA is likely wound around the L1ORF1p trunk with the strand passing through the negatively charged vertical and horizontal grooves. So far, however, it is impossible to experimentally verify the structure of the RNA-protein interactions. | ||
| + | |||
| + | ===Structural Flexibility=== | ||
| + | The CTD domains are "hinged" to the RRM and coiled-coil domains, and are free to move relative to the rest of the structure. This might be important for reverse transcription, allowing the trimer to unwind the RNA slowly so as to prevent the formation of secondary structure in the transcript. There was found a <scene name='57/579701/Orf1p_overall_structure/13'>"closed" conformation</scene> and an <scene name='57/579701/Orf1p_open_structure/1'>"open"</scene> one. | ||
| + | |||
| + | ==3D Structures of L1 ORF1 Protein== | ||
| + | |||
| + | ===Mus musculus (mouse)=== | ||
| + | Nuclear Magnetic Resonance: 2JRB, 2LDY, 2W7A | ||
| + | |||
| + | ===Homo sapiens (human)=== | ||
| + | X-Ray Crystallography: 2YKO, 2YKP, 2YKQ | ||
| + | |||
| + | ==See Also== | ||
| + | [http://www.nature.com/nsmb/journal/v18/n9/extref/nsmb.2097-S2.mov Movie of conformational change] | ||
| + | |||
| + | ==References== | ||
| + | {{reflist}} | ||
Current revision
| This Sandbox is Reserved from 01/04/2014, through 30/06/2014 for use in the course "510042. Protein structure, function and folding" taught by Prof Adrian Goldman, Tommi Kajander, Taru Meri, Konstantin Kogan and Juho Kellosalo at the University of Helsinki. This reservation includes Sandbox Reserved 923 through Sandbox Reserved 947. |
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LINE-1 ORF1 Protein
Long interspersed nuclear element 1 (LINE1) is a non-long terminal repeat (non-LTR) type retrotransposon in mammals that engages in retrotransposition - a process which randomly inserts LINE1's own coding sequence into the host genome, increasing the genome size and causing genomic instability. The LINE1 gene encodes the open reading frame 1 protein (L1ORF1p), which localizes to large L1 ribonucleoprotein particles, stress granules and nucleus (ref), and is required and necessary for retrotransposition.
Introduction
After transcription of the LINE1 gene, the same RNA transcript is both translated and is the template for reverse transcription. This RNA transcript contains two open reading frames: ORF1 and ORF2, which codes for two proteins, ORF1p and ORF2p. L1ORF1p contains the RNA packing and delivery functions, and L1ORF2p contains the enzymatic machinery for reverse transcription. Translation of both of these open reading frames is necessary for retrotransposition. The reverse transcribed LINE1 is integrated into the host genome in a different place than the original gene.
L1ORF1p localizes to ribonucleoprotein particles, stress granules and nucleus. Although the protein has general affinity to nucleic acids, it displays a strong cis preference, which makes it bind the primary encoding RNA transcript[1]. The role of L1ORF1p in retrotransposition is to protect the transcript from degradation and to help transport it to the nucleus.
Structure
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