Part:BBa_K2624002
Nuclear localized NS5B
Nonstructural protein 5B (NS5B) is a viral protein found in the hepatitis C virus (HCV). It is a RNA-dependent RNA polymerase and initiates de novo RNA synthesis with the help of its specific RNA promoters. If you need to generate double stranded RNAs, or substitutional hybridization in situ in the nucleus, this is probably what you are looking for.
Usage
The 2018 CPU_CHINA project is a gene therapy strategy based on conditional RNA interference. The RNA interference is conditional because the pri-miRNA analogue partially paired with an inhibitory stranded is unable to be processed by Drosha. Once a RNA-dependent RNA polymerase (like this one) is introduced to catalyze a substitutional hybridation to release the pri-miRNA in the nucleus, the analogue get to evolve into miRNA and interfere with the target gene product.
Biology
RNA-dependent RNA polymerase (RdRP) is an enzyme that catalyzes the replication of RNA from an RNA template. It is quite an essential protein for all RNA-containing viruses with no DNA stage i.e. only RNA viruses. With a positive sense single-stranded RNA as its genome, Hepatitis C virus is such a virus. The corresponding RdRP is the nonstructural protein 5B (NS5B) of the HCV polyprotein. Human cells do not have proteins similar in function.
HCV NS5B belongs to a class of membrane proteins termed “tail-anchored proteins”, which feature posttranslational membrane targeting via a C-terminal transmembrane domain (TMD). The polymerase’s catalytic activity is independent of this TMD, so recombinant soluble NS5B usually does not contain the last 21 or 51 amino acids at the C-terminal.
The structure of HCV NS5B can be represented by a right hand shape with fingers, palm, and thumb. The encircled active site, unique to NS5B, is contained within the palm structure of the protein. It initiates RNA synthesis with nucleotide transfer activity found within the palm motif, with also several amino acid residues implicated in nucleotide triphosphate contact. Kao et al. discovered specific template requirements for de novo initiation of this polymerase. All the RNA promoters they identified possess a cytidylate at or near the 3’ terminus that could act as a potential initiation site for RNA synthesis. A ladder of polymerized products is expected because along with many well-characterized polymerases, NS5B has the property of nontemplate nucleotide addition.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 492
Illegal BglII site found at 1340 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 537
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