Author: te Velthuis, Aartjan J.W.; van den Worm, Sjoerd H. E.; Snijder, Eric J.
Title: The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension Document date: 2011_10_29
ID: tx0lqgff_26
Snippet: A unique feature of the hexadecameric SARS-CoV nsp(7+8) structure is the fact that it does not derive from stacking of its protein subunits, but rather from stable inter-connections of the 'golf club-like' nsp8 molecules ( Figure 1B) (13) . The structural support of the nsp8 , followed by purification and cleavage by recombinant SARS-CoV nsp5 main protease to remove the C-terminal His 6 -tag and its upstream GSSG linker. (B) Eighteen percent SDS-.....
Document: A unique feature of the hexadecameric SARS-CoV nsp(7+8) structure is the fact that it does not derive from stacking of its protein subunits, but rather from stable inter-connections of the 'golf club-like' nsp8 molecules ( Figure 1B) (13) . The structural support of the nsp8 , followed by purification and cleavage by recombinant SARS-CoV nsp5 main protease to remove the C-terminal His 6 -tag and its upstream GSSG linker. (B) Eighteen percent SDS-PAGE analysis of nsp5-treated, purified nsp8-His demonstrates near-complete release of the C-terminal His 6 -tag within 60 min. The maltose binding protein (MBP) was added to the reaction to serve as an independent loading control. Asterisks indicate non-specific bands. (C) In addition to the tag-less nps8 and nsp8-His, we also produced the N-terminally His 6 -tagged nsp8 (His-nsp8) used by Imbert et al. (12) . (D) Comparative gel filtration analysis of nsp8 (22 kDa as a monomer) versus His-nsp8 and (E) nsp8 versus nsp8-His. In all three cases, nsp8 formed multimers in solution, but the apparent molecular mass of complexes formed by both nsp8 and nsp8-His was $2-fold higher than for complexes formed by His-nsp8. (F) Comparative analysis of nsp8, nsp(7+8), His-nsp8 and nsp7+nsp8-His. Only nsp(7+8) showed a molecular weight shift to the $225-kDa size range with a standard deviation of 15-kDa (n = 3). This size is indicative of hexadecamer formation, whereas the analysis of nsp7+nsp8-His showed dominant peaks of nsp8-His and nsp7 (which is $10 kDa as a monomer). octamer by eight copies of nsp7 thus appears to be redundant, in line with the critical role for the nsp8 N-terminal domain described above. We surmised therefore that the additional complexity must have evolved to improve nsp8's function and set out to compare the RNA binding capabilities of the purified nsp8 octamer and nsp(7+8) hexadecamer.
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