Sandbox Reserved 1085

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The <scene name='69/699998/Monomer/2'>monomer</scene> of Aquifex aeolicus RNase III (Aa-RNase III) are composed of an <scene name='69/699998/Endond/1'>endonuclease domain</scene> (endoND, purple) and a <scene name='69/699998/Dsrbd/3'>dsRNA binding domain</scene> (dsRBD, red)<ref>Lamontagne, B., et al., The RNase III family: a conserved structure and expanding functions in eukaryotic dsRNA metabolism. Yeast, 2001. 45(191): p. 154-158.</ref>. The sequence of the endoND is characterized by a stretch of conserved residues (37ERLEFLGD44 in Aa-RNase III), which is known as the RNase III signature motif and makes up a large part of the active center. RNase III can affect gene expression in either of two ways: as a processing enzyme which RNase III cleaves both natural and synthetic dsRNA into small duplex products averaging 10–18 base pairs in length, or as a binding protein which binds and stabilizes certain RNAs, thus suppressing the expression of certain genes[<ref>Robertson, H.D., Escherichia coli ribonuclease III cleavage sites. Cell, 1982. 30(3): p. 669-672.</ref>, <ref>Grunberg-Manago, M., Messenger RNA stability and its role in control of gene expression in bacteria and phages. Annual review of genetics, 1999. 33(1): p. 193-227.</ref>].
The <scene name='69/699998/Monomer/2'>monomer</scene> of Aquifex aeolicus RNase III (Aa-RNase III) are composed of an <scene name='69/699998/Endond/1'>endonuclease domain</scene> (endoND, purple) and a <scene name='69/699998/Dsrbd/3'>dsRNA binding domain</scene> (dsRBD, red)<ref>Lamontagne, B., et al., The RNase III family: a conserved structure and expanding functions in eukaryotic dsRNA metabolism. Yeast, 2001. 45(191): p. 154-158.</ref>. The sequence of the endoND is characterized by a stretch of conserved residues (37ERLEFLGD44 in Aa-RNase III), which is known as the RNase III signature motif and makes up a large part of the active center. RNase III can affect gene expression in either of two ways: as a processing enzyme which RNase III cleaves both natural and synthetic dsRNA into small duplex products averaging 10–18 base pairs in length, or as a binding protein which binds and stabilizes certain RNAs, thus suppressing the expression of certain genes[<ref>Robertson, H.D., Escherichia coli ribonuclease III cleavage sites. Cell, 1982. 30(3): p. 669-672.</ref>, <ref>Grunberg-Manago, M., Messenger RNA stability and its role in control of gene expression in bacteria and phages. Annual review of genetics, 1999. 33(1): p. 193-227.</ref>].
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[[Image: Binding site 1.png|thumb|left|420px|Fig.1. ''RNase III (Pbd codes 1O0W & 2EZ6 superimposition using Pymol'' ]]]
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[[Image: Binding site 1.png|thumb|left|420px|Fig.1. ''RNase III (Pbd codes 1O0W & 2EZ6 superimposition using Pymol'' ]]
On the basis of the structural and biochemical data, catalytic models were proposed before the structure of a catalytic complex became available. The crystal structure shows that Aa-RNase III is composed of a <scene name='69/699998/Dimer/3'>symmetric dimer</scene>. In addition, in vivo data suggested that E110, E37, D44, and E64 are essential for catalysis<ref>Blaszczyk, J., et al., Noncatalytic assembly of ribonuclease III with double-stranded RNA. Structure, 2004. 12(3): p. 457-466.</ref>. This led to the model of the proteins active centers, which can accommodate a dsRNA substrate, each containing two different RNA cleavage sites, <scene name='69/699998/D44_e110_e37_e64/1'>D44/E110 and E37/E64</scene> (pink and red). Specifically, E64 from each partner subunit, along with E37, E40, and D44 are located in the signature motif located at each end of the catalytic valley<ref>Blaszczyk, J., et al., Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage. Structure, 2001. 9(12): p. 1225-1236</ref>. Comparing the structure of Aa-RNase III with the structure of RNA-free Thermotoga maritima RNase III (RNA-free Tm-RNase III, PDB ID code 1O0W)<ref>Gan, J., et al., Intermediate states of ribonuclease III in complex with double-stranded RNA. Structure, 2005. 13(10): p. 1435-1442.</ref> shows that there is <scene name='69/699998/Linker_rotation/1'>dramatic rotation</scene> and shift of dsRBD due to RNA binding, and there is a <scene name='69/699998/Linker/2'>flexible linker</scene> KGEMLFD between endoRD and dsRBD leading the rotation happend. In addtion, the two dsRBDs are apart from each other, allowing free rotation of dsRBDdsRNA around the linker.
On the basis of the structural and biochemical data, catalytic models were proposed before the structure of a catalytic complex became available. The crystal structure shows that Aa-RNase III is composed of a <scene name='69/699998/Dimer/3'>symmetric dimer</scene>. In addition, in vivo data suggested that E110, E37, D44, and E64 are essential for catalysis<ref>Blaszczyk, J., et al., Noncatalytic assembly of ribonuclease III with double-stranded RNA. Structure, 2004. 12(3): p. 457-466.</ref>. This led to the model of the proteins active centers, which can accommodate a dsRNA substrate, each containing two different RNA cleavage sites, <scene name='69/699998/D44_e110_e37_e64/1'>D44/E110 and E37/E64</scene> (pink and red). Specifically, E64 from each partner subunit, along with E37, E40, and D44 are located in the signature motif located at each end of the catalytic valley<ref>Blaszczyk, J., et al., Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage. Structure, 2001. 9(12): p. 1225-1236</ref>. Comparing the structure of Aa-RNase III with the structure of RNA-free Thermotoga maritima RNase III (RNA-free Tm-RNase III, PDB ID code 1O0W)<ref>Gan, J., et al., Intermediate states of ribonuclease III in complex with double-stranded RNA. Structure, 2005. 13(10): p. 1435-1442.</ref> shows that there is <scene name='69/699998/Linker_rotation/1'>dramatic rotation</scene> and shift of dsRBD due to RNA binding, and there is a <scene name='69/699998/Linker/2'>flexible linker</scene> KGEMLFD between endoRD and dsRBD leading the rotation happend. In addtion, the two dsRBDs are apart from each other, allowing free rotation of dsRBDdsRNA around the linker.
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[[Image: ALIGN2.png|thumb|right|420px|Fig.2. ''RNase III (Pbd codes 1O0W & 2EZ6 superimposition using Pymol'' ]]]
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[[Image: ALIGN2.png|thumb|right|420px|Fig.2. ''RNase III (Pbd codes 1O0W & 2EZ6 superimposition using Pymol'' ]]
== A Hypothetical Pathway Leading to the two Functional Forms of RNase III ==
== A Hypothetical Pathway Leading to the two Functional Forms of RNase III ==
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Catalytic processes in addition to the catalytic and noncatalytic forms of the Aa-RNase III–dsRNA structures revealed possible intermediate states of dsRNA binding and a hypothetical pathway of RNase III in vivo. The RNA-free RNase III dimer in conformation A (Figure a, PDB 1O0W) first binds a dsRNA with one of the two dsRBDs. The resulting complex may have at least two possible conformations, B (Figure b, PDB1YZ9 AaRNase IIIE110Q) and E (Figure e, PDB 1YYO), in which the dsRNA is located outside of the catalytic valley. These two conformations, b and e, appear to be interchangeable. In conformation E, the two dsRBDs pack against each other, hindering free rotation of the dsRBD-dsRNA complex around the flexible linker between the endoND and dsRBD. Conformation b allows free rotation of dsRBD-dsRNA around the linker (Figure b). The clockwise rotation of dsRBD-dsRNA leads to the catalytic form (Figure d, PDB 2EZ6) via conformation C (Figure c, PDB 1YYW). Conformations B and C are also likely to be interchangeable. In contrast, a factor that uncouples binding and processing can further stabilize conformation E leading to the noncatalytic form of RNase III (Figure f, PDB 1RC7)[<ref>Blaszczyk, J., et al., Noncatalytic assembly of ribonuclease III with double-stranded RNA. Structure, 2004. 12(3): p. 457-466</ref>, <ref>Gan, J., et al., Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III. Cell, 2006. 124(2): p. 355-66</ref>, <ref>Gan, J., et al., Intermediate states of ribonuclease III in complex with double-stranded RNA. Structure, 2005. 13(10): p. 1435-1442.</ref>].
Catalytic processes in addition to the catalytic and noncatalytic forms of the Aa-RNase III–dsRNA structures revealed possible intermediate states of dsRNA binding and a hypothetical pathway of RNase III in vivo. The RNA-free RNase III dimer in conformation A (Figure a, PDB 1O0W) first binds a dsRNA with one of the two dsRBDs. The resulting complex may have at least two possible conformations, B (Figure b, PDB1YZ9 AaRNase IIIE110Q) and E (Figure e, PDB 1YYO), in which the dsRNA is located outside of the catalytic valley. These two conformations, b and e, appear to be interchangeable. In conformation E, the two dsRBDs pack against each other, hindering free rotation of the dsRBD-dsRNA complex around the flexible linker between the endoND and dsRBD. Conformation b allows free rotation of dsRBD-dsRNA around the linker (Figure b). The clockwise rotation of dsRBD-dsRNA leads to the catalytic form (Figure d, PDB 2EZ6) via conformation C (Figure c, PDB 1YYW). Conformations B and C are also likely to be interchangeable. In contrast, a factor that uncouples binding and processing can further stabilize conformation E leading to the noncatalytic form of RNase III (Figure f, PDB 1RC7)[<ref>Blaszczyk, J., et al., Noncatalytic assembly of ribonuclease III with double-stranded RNA. Structure, 2004. 12(3): p. 457-466</ref>, <ref>Gan, J., et al., Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III. Cell, 2006. 124(2): p. 355-66</ref>, <ref>Gan, J., et al., Intermediate states of ribonuclease III in complex with double-stranded RNA. Structure, 2005. 13(10): p. 1435-1442.</ref>].
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[[Image: Ge470405.f7.gif|thumb|left|420px|Fig.2. ''Hypothetical Pathway Leading to the two Functional Forms of RNase III'' ]]]
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[[Image: Ge470405.f7.gif|thumb|left|840px|Fig.2. ''Hypothetical Pathway Leading to the two Functional Forms of RNase III'' ]]

Revision as of 15:40, 22 April 2015

This Sandbox is Reserved from 15/04/2015, through 15/06/2015 for use in the course "Protein structure, function and folding" taught by Taru Meri at the University of Helsinki. This reservation includes Sandbox Reserved 1081 through Sandbox Reserved 1090.
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Endoribonuclease III

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References

  1. ↑ Lioliou E, Sharma CM, Caldelari I, et al. Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression. Hughes D, ed. PLoS Genetics. 2012;8(6):e1002782. doi:10.1371/journal.pgen.1002782
  2. ↑ Lamontagne, B., et al., The RNase III family: a conserved structure and expanding functions in eukaryotic dsRNA metabolism. Yeast, 2001. 45(191): p. 154-158.
  3. ↑ Robertson, H.D., Escherichia coli ribonuclease III cleavage sites. Cell, 1982. 30(3): p. 669-672.
  4. ↑ Grunberg-Manago, M., Messenger RNA stability and its role in control of gene expression in bacteria and phages. Annual review of genetics, 1999. 33(1): p. 193-227.
  5. ↑ Blaszczyk, J., et al., Noncatalytic assembly of ribonuclease III with double-stranded RNA. Structure, 2004. 12(3): p. 457-466.
  6. ↑ Blaszczyk, J., et al., Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage. Structure, 2001. 9(12): p. 1225-1236
  7. ↑ Gan, J., et al., Intermediate states of ribonuclease III in complex with double-stranded RNA. Structure, 2005. 13(10): p. 1435-1442.
  8. ↑ Blaszczyk, J., et al., Noncatalytic assembly of ribonuclease III with double-stranded RNA. Structure, 2004. 12(3): p. 457-466
  9. ↑ Gan, J., et al., Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III. Cell, 2006. 124(2): p. 355-66
  10. ↑ Gan, J., et al., Intermediate states of ribonuclease III in complex with double-stranded RNA. Structure, 2005. 13(10): p. 1435-1442.
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