NS5B: Difference between revisions
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<scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA. | <scene name='NS5B/Native_ns5b/6'>beta-hairpin in thumb domain</scene> that is proposed to move upon formation of exiting double stranded RNA. | ||
<applet load='2HAI_DNA5.pdb' size=' | <applet load='2HAI_DNA5.pdb' size='400' frame='true' align='left' caption='Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA)' />'''Figure 2''' is a model of NS5B with B form DNA. DNA was modeled into the NS5B model by aligning of palm domain of NS5B and the palm domain of HIV reverse transcriptase, which was co-crystallized in complex with DNA and an incoming dTTP. Then removing the protein portion HIV RT model while leaving the DNA where it fell into the proposed NS5B binding cleft. Looking closely at the <scene name='NS5B/Ns5b_with_dna/3'>active site</scene> the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A <scene name='NS5B/Ns5b_with_dna/4'>beta-hairpin</scene> (residues 440-455) in the thumb domain has been shifted to accommodate DNA, the hairpin is modeled into the minor groove, a possible binding site, particularly in the larger minor goove of dsRNA. There are noticeable steric clashes between the modeled DNA and the random coil at the end of the c-terminal domain. This domain is a linker that attaches to the membrane anchor of NS5B. | ||
The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains. | The template strand is seen entering through a gap in the fingers domain. An incoming dTTP that would extend the primer strand lines up with the NS5B active site and duplex DNA exits the enzyme through the large central hole in the closed active site formed by the unusual contacts between the fingers and thumb domains. | ||
<applet load='2HAI_catalytic3.pdb' size=' | <applet load='2HAI_catalytic3.pdb' size='700' frame='true' align='right' caption='Figure 3.' /> | ||
'''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. | '''Figure 3''' explores empirically determined sites of protein-ssRNA interactions. The highlighted <scene name='NS5B/Ns5b_rna_interactions/1'>peptide segments</scene> were each identified without x-ray crystallography or NMR. These RNA binding peptides were identified by cross linking single stranded RNA to NS5B followed by a tryptic digest of the protein, then purification of the RNA bound peptide segments by affinity (for the RNA) chromatography. The segments of peptide that stuck to the column meaning they had been cross linked to RNA were then analyzed with MALDI mass spectrometry. It is interesting that all of the contacts were in the <scene name='NS5B/Native_ns5b/3'>fingers domain.</scene> This could be in part due to the fact that single stranded RNA was cross linked to the enzyme, the fingers domain is thought to bind templating ssRNA while other regions of the polymerase would bind duplex RNA. | ||
