Part:BBa_K1442103

Reverse 5

Each unique RNA dependent RNA polymerase (RdRP) initiates de novo replication of a RNA strand by interacting with RdRP-specific RNA sequences, henceforth called RdRP/RNA promoters. Please refer to Section: RdRP function for further details. The RdRP chosen for our project is taken from the Hepatitis C virus (HCV). It starts replication at the 3’ end of the HCV RNA strand. This 3’ UTR is a RNA promoter. After producing the minus strand of the HCV RNA, the RdRP “recognises” the reversed 5’UTR of the original RNA and initiates second replication which produces the viral RNA in is positive sense . Therefore, the reversed 5’ UTR (R5) of HCV is also a RNA promoter. The complete sequence was taken from the NCBI GenBank . It has been suggested that the first 125 nucleotides of the 5’UTR are sufficient for replication, but the inclusion of the entire 341 base-long region results in higher efficiency rates . Due to the fact that the 5’UTR and a part of the coding sequence constitute an IRES essential for the translation of the HCV, another 76 nucleotides from the coding DNA were included in the sequence for the promoter.

Usage

Used as an RNA promoter to direct replication by RdRP.

RdRP-directed Replication

1. The DNA strand gets transcribed and then translated, whereby the RdRP protein is produced.

2. The active RdRP binds to the 3’ end of the transcribed plus –sense RNA strand. It produces its complementary minus-sense RNA strand.

3. The RdRP interacts with the 5’ end of the replicated minus-sense RNA strand. It produces the plus-sense RNA strand again and completes the full replication of the RNA. The new RNA strand can act as a template as well and the process of replication can start over.

Mathematical Modelling

E-coli

Where c, α-,β are constants, E stands for the RdRP, G – for GFP. R- is amount of minus-sense RNA strands, R+ amount of plus-sense RNA strands; µ- is degradation rate of the minus-sense strands and µ+of the plus-sense.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 142
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 397
  Illegal NgoMIV site found at 426
  Illegal AgeI site found at 236
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 69