Team:Uppsala University/Modelling
From 2012.igem.org
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The wildtype small RNA of E.coli K-12 MG1655 that we used as a template for constructing our own smallRNA is named Spot42 and has been shown to be interacting with Hfq pro | The wildtype small RNA of E.coli K-12 MG1655 that we used as a template for constructing our own smallRNA is named Spot42 and has been shown to be interacting with Hfq pro | ||
</p> | </p> | ||
+ | <br> | ||
<p> | <p> | ||
Spot42 has like many other smallRNA, two distinctive parts. One that binds to a mRNA sequence, and another sequence that interacts with the hfq-rna binding protein that is belived to play an important role in the function of smallRNA. (Holmquist, 2012) | Spot42 has like many other smallRNA, two distinctive parts. One that binds to a mRNA sequence, and another sequence that interacts with the hfq-rna binding protein that is belived to play an important role in the function of smallRNA. (Holmquist, 2012) | ||
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To gain better understanding of the mechanisms of smallRNA translation inhibition we have made 2D models of all our found smallRNAs. The 2D models were calculated in CLC main workbench.<br> | To gain better understanding of the mechanisms of smallRNA translation inhibition we have made 2D models of all our found smallRNAs. The 2D models were calculated in CLC main workbench.<br> | ||
IntaRNA, an RNA-interaction prediction software adapted for sRNA and ncRNA interactions (Smith et. al., 2010) was used to predict the sRNA-mRNA interactions of the candidate sRNAs. | IntaRNA, an RNA-interaction prediction software adapted for sRNA and ncRNA interactions (Smith et. al., 2010) was used to predict the sRNA-mRNA interactions of the candidate sRNAs. | ||
+ | </p> | ||
+ | <br> | ||
+ | <p> | ||
+ | The sRNA were isolated, sequenced and analyzed to find the hybridizing base pairs. The sequencing of the sRNAs that showed downregulation of YFP also had a matching sequence in the 5’UTR of our target mRNA.<br> | ||
+ | Altough, a few of the sRNA that downregulated YFP was shown to hybridize at the YFP mRNA region. Two of these were further studied and modelled. | ||
+ | At last, a prediction of the structure between the sRNA UU17 and AAC(6’)UTR mRNA were modelled. | ||
+ | </p> | ||
+ | <br> | ||
+ | <p> | ||
+ | As you can see below, our sRNA that downregulates the YFP and the antibiotic resistance gene also shows to hybridize at the region close to the RBS and the start codon. This supports the idea that many sRNA prevents the ribosome from binding to the RBS, and thereby preventing translation. (Erik Holmquist, 2012)<br> | ||
+ | Also our predictions data from clc shows that the hybridizing area have a strong secondary structure, with small hairpin loops. | ||
+ | </p> | ||
+ | <br> | ||
+ | <p> | ||
+ | [nBp] = Total number of base pairs in sRNA | ||
+ | [nBpHyb] = Number of bases hybridizing (calculated from freiburg) intaR | ||
+ | [dG] = delta G; Predicted free energy for secondary structure and mRNA-sRNA interaction. | ||
+ | [nInRowBp] = Maximum number of hybridizing bases in a row | ||
</p> | </p> | ||
<br> | <br> | ||
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<img src="https://static.igem.org/mediawiki/2012/f/f8/SRNA17structure_medium.png" width=150> | <img src="https://static.igem.org/mediawiki/2012/f/f8/SRNA17structure_medium.png" width=150> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <b>sRNA UU37</b><br> | ||
+ | <table id="ttable"> | ||
+ | <tr> | ||
+ | <td> | ||
+ | [dG] = -31.9 kcal/mol | ||
+ | </td> | ||
+ | <td> | ||
+ | [nBp] = 83<br> | ||
+ | [nBpHyb] = 24<br> | ||
+ | [nInRowBp] = 7<br> | ||
+ | [Etest2409]= 64<br> | ||
+ | [dG]=<br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <img src="https://static.igem.org/mediawiki/2012/" width=150> | ||
+ | </td> | ||
+ | <td> | ||
+ | <img src="https://static.igem.org/mediawiki/2012/" width=150> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <b>sRNA UU46</b><br> | ||
+ | <table id="ttable"> | ||
+ | <tr> | ||
+ | <td> | ||
+ | [dG] = -28.7 kcal/mol<br> | ||
+ | </td> | ||
+ | <td> | ||
+ | [dG] = <br> | ||
+ | [nBp] = 65 bp<br> | ||
+ | [nBpHyb] = 8 bp<br> | ||
+ | [nInRowBp] = 8 bp<br> | ||
+ | [Etest2409]=Not readable<br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <img src="https://static.igem.org/mediawiki/2012/" width=150> | ||
+ | </td> | ||
+ | <td> | ||
+ | <img src="https://static.igem.org/mediawiki/2012/" width=150> | ||
</td> | </td> | ||
</tr> | </tr> |
Revision as of 18:01, 25 September 2012
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