NS5B

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	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.		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.
-	Figure 4. [[Image:PolyProtein.jpg]]	+	[[Image:PolyProtein.jpg]]
	http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html		http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html
		+	Figure 4.
	== '''NS5B + LIGANDS''' ==		== '''NS5B + LIGANDS''' ==

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Revision as of 03:10, 21 April 2009

RNA Dependent RNA Polymerase from Hepatitis C Virus

spinqualitylabelsall models Figure 1. PDB ID 2HAI Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

NS5B is the RNA dependent RNA polymerase of Hepatitis C virus. NS5B, like other RNA dependent RNA polymerases, is error prone, this viral RNA replicase is of approximately a million times lower fidelity than a replicative prokayrotic or eukaryotic DNA polymerase. This is due in part to the fact that NS5B contains no exonuclease or proofreading domain. The proposed mechanism for NS5B polymerization of monomers of RNA triphosphates to extend a primer strand, that may have initiated de novo, is via two divalent cations coordinated by carboxyl groups as seen in DNA polymerases. In the case of NS5B the residues that coordinate divalent cations (Mg2+ or Mn2+ in vitro) are the three

seen here.

Though Hepatitis C virus is of the Flaviviridae family the structure of NS5B is similar to the polymerase of bacteriophage Φ 6. The similarity to the bacteriophage polymerase is due to NS5B containing a fully encircled active site. Like many template-dependent nucleotide polymerases, NS5B can be visualized similar to a right hand. NS5B contains several , fingers in blue, palm in magenta, thumb in green and a c-terminal domain in yellow. The palm domain contains the active site aspartates and there are several contacts between the fingers and thumbs domain that give the active site an encircled structure. There is a that is proposed to move upon formation of exiting double stranded RNA.

spinqualitylabelsall models Figure 2. PDB IDs 2HAI (protein) 1RTD (DNA) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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 the catalytic Mg2+ ions are modeled in green, these would be coordinated by the three aspartic acid carboxylates, (D220, D318 and D319). A (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 where many of the deposited NS5B structures are disordered or have high b-factors so it is likely that the protein is mobile in this region and can thus accommodate RNA.

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.

spinqualitylabelsall models Figure 3. Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Figure 3 explores empirically determined sites of protein-ssRNA interactions. The highlighted 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 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.

Image:PolyProtein.jpg

http://www.nature.com/nrmicro/journal/v5/n6/fig_tab/nrmicro1645_F4.html Figure 4.

NS5B + LIGANDS

spinqualitylabelsall models Figure 5. NS5B in complex with ligands from deposited structures all superimposed on the 1.58Å resolution 2HAI. Ligands are from Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image