Difference between revisions of "Part:BBa K1442118:Design"
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<partinfo>BBa_K1442118 short</partinfo> | <partinfo>BBa_K1442118 short</partinfo> | ||
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<partinfo>BBa_K1442118 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1442118 SequenceAndFeatures</partinfo> | ||
| + | == RNA and secondary structure == | ||
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| + | [[File:5'.gif]] | ||
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| + | == Ribozyme == | ||
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The use of a ribozyme is necessitated due to the complicated binding process between the RNA template and the RdRP. In order to optimise the process and avoid any risk of unfavourable secondary structures or obstruction by unneeded nucleotides, a self-cleaving ribozyme was put after the RNA promoter. The rationale being that after transcription (either in vitro for human cell tests or in vivo for e-coli tests), any added bases as a result of the T7 polymerase would be removed from the main template strand. The chosen ribozyme taken from the Hepatitis Delta virus was investigated at length by the team of J. Doudna and is reported to be the fasted naturally occurring, independent and resistant to denaturants. Its close genetic origin also contribute to a better working and compatible system. | The use of a ribozyme is necessitated due to the complicated binding process between the RNA template and the RdRP. In order to optimise the process and avoid any risk of unfavourable secondary structures or obstruction by unneeded nucleotides, a self-cleaving ribozyme was put after the RNA promoter. The rationale being that after transcription (either in vitro for human cell tests or in vivo for e-coli tests), any added bases as a result of the T7 polymerase would be removed from the main template strand. The chosen ribozyme taken from the Hepatitis Delta virus was investigated at length by the team of J. Doudna and is reported to be the fasted naturally occurring, independent and resistant to denaturants. Its close genetic origin also contribute to a better working and compatible system. | ||
| + | [[File:Rib1.gif]] | ||
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| + | The chosen ribozyme taken from the Hepatitis Delta virus was investigated at length by the team of J. Doudna and is reported to be the fastest naturally occurring self-cleaving protein. It also functions independently, without the need for adding chemical substances, and is resistant to denaturants. Its close genetic origin to the RdRP also contributes to a better working and more compatible system. | ||
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| + | The structure of the molecule and its active site in particular are shown below. | ||
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| + | [[File:Rib2.gif]] | ||
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Hepatitis C Virus | Hepatitis C Virus | ||
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| + | “Partial reconstitution of hepatitis C virus RNA polymerization by heterologous expression of NS5B polymerase and template RNA in bacterial cell”, 2005, Sangyoon Lee, Jong-Ho Lee, Young Hoon Kee, Mi Young Park, Heejoon Myung | ||
| + | |||
| + | B. Heinz, C. Kao – “Template Requirements for RNA Synthesis by a Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase” - [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC113194/] | ||
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| + | “Hepatitis C virus type 1b complete genome, isolate Con1”- [http://www.ncbi.nlm.nih.gov/nuccore/AJ238799] | ||
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| + | “Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line”, 1999, V. Lohmann, F. Körner, J.-O. Koch, U. Herian, L. Theilmann, R. Bartenschlager | ||
| + | |||
| + | “Hepatitis C virus subtype 1b isolate 31 polyprotein gene, partial cds”- | ||
| + | [http://www.ncbi.nlm.nih.gov/nuccore/KJ679443.1] | ||
| + | |||
| + | “Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication”, 2001, Peter Friebe, Volker Lohmann, Nicole Krieger, and Ralf Bartenschlager | ||
| + | |||
| + | “Crystal structure of a hepatitis delta virus ribozyme”, 1998, Adrian R. Ferré-D'Amaré, Kaihong Zhou and Jennifer A. Doudna | ||
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===References=== | ===References=== | ||
Latest revision as of 22:52, 16 October 2014
5' RdRP Promoter
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 292
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 198
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 365
RNA and secondary structure
Ribozyme
The use of a ribozyme is necessitated due to the complicated binding process between the RNA template and the RdRP. In order to optimise the process and avoid any risk of unfavourable secondary structures or obstruction by unneeded nucleotides, a self-cleaving ribozyme was put after the RNA promoter. The rationale being that after transcription (either in vitro for human cell tests or in vivo for e-coli tests), any added bases as a result of the T7 polymerase would be removed from the main template strand. The chosen ribozyme taken from the Hepatitis Delta virus was investigated at length by the team of J. Doudna and is reported to be the fasted naturally occurring, independent and resistant to denaturants. Its close genetic origin also contribute to a better working and compatible system.
The chosen ribozyme taken from the Hepatitis Delta virus was investigated at length by the team of J. Doudna and is reported to be the fastest naturally occurring self-cleaving protein. It also functions independently, without the need for adding chemical substances, and is resistant to denaturants. Its close genetic origin to the RdRP also contributes to a better working and more compatible system.
The structure of the molecule and its active site in particular are shown below.
Source
Hepatitis C Virus
“Partial reconstitution of hepatitis C virus RNA polymerization by heterologous expression of NS5B polymerase and template RNA in bacterial cell”, 2005, Sangyoon Lee, Jong-Ho Lee, Young Hoon Kee, Mi Young Park, Heejoon Myung
B. Heinz, C. Kao – “Template Requirements for RNA Synthesis by a Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase” - [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC113194/]
“Hepatitis C virus type 1b complete genome, isolate Con1”- [http://www.ncbi.nlm.nih.gov/nuccore/AJ238799]
“Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line”, 1999, V. Lohmann, F. Körner, J.-O. Koch, U. Herian, L. Theilmann, R. Bartenschlager
“Hepatitis C virus subtype 1b isolate 31 polyprotein gene, partial cds”- [http://www.ncbi.nlm.nih.gov/nuccore/KJ679443.1]
“Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication”, 2001, Peter Friebe, Volker Lohmann, Nicole Krieger, and Ralf Bartenschlager
“Crystal structure of a hepatitis delta virus ribozyme”, 1998, Adrian R. Ferré-D'Amaré, Kaihong Zhou and Jennifer A. Doudna