Artificial transcriptional terminators

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The goal is to create a series of transcriptional terminators with varying efficiencies. The majority of transcriptional terminators have a G+C rich stem of 7(+/-1)bp and a loop of 4(+/-1) nucleodtides followed by a poly(U) tail. Two common loops are UUCG and GAAA, both of which are known to increase RNA hairpin stability. The sequence GCGGG(G) is a common sequence found on the 3' arm of the stem. [d'Aubenton 90]

  • Effects of stem loop sequence on terminator efficiency

Bulges and mismatches in the stem, as well low G+C content of the stem will lower TE more than reducing the length of or elimination of the poly(U) tail [Abe 96]. The sequences downstream of the poly(U) tail and between the stop codon and the start of the stem loop structure also affect the TE of a terminator, particularly T7Te or T3Te.

  • T7Te

Several sources [Reynolds 92, Chamberlin 79] measured the termination efficiency(TE) of T7Te at around 90%. However, efficiency for the biobricks part BBa_B0012 [1], also T7Te, is around 30%. T7Te has a very short poly(U) tail and requires the further downstream sequence for efficiecent termination [Reynolds 92], and this further downstream sequence is lacking in BBa_B0012. If the sequence for BBa_B0012 is lengthened to include this downstream segment, then the TE of part should be improved.

  • Predicting terminator efficiency

It may be possible to predict terminator efficiency using methods from d'Aubenton, in particular, the score d assigned to a possible terminator sequence

''d'' = nt * 18.16 + Y * 96.59 − 116.87 where nt measures the statistical distribution of the T residues in the non transcribed DNA strand and Y is the free energy per nucleodtide of the stem loop structure.

The score d will give a rough estimate of how efficient a terminator is. d<0: TE<20% 0<d<30: 20%<TE<70% d>30: TE>70%

References

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  1. Error fetching PMID 9150882: [Abe96]
  2. Error fetching PMID 1702475: [Aubenton90]
  3. Error fetching PMID 3078109: [Bredel86]
  4. Error fetching PMID 1835546: [Cheng91]
  5. Error fetching PMID 7027254: [Christie81]
  6. Error fetching PMID 10926490: [Ermolaeva00]
  7. Error fetching PMID 11522828: [Lesnik01]
  8. Error fetching PMID 2961747: [Lynn88]
  9. Error fetching PMID 16820051: [Petrillo06]
  10. Error fetching PMID 1372365: [Reynolds92I]
  11. Error fetching PMID 1372366: [Reynolds92II]
  12. Error fetching PMID 11809879: [Unniraman02]
  13. Error fetching PMID 9391063: [Uptain97]
  14. Error fetching PMID 1536005: [VonHippel92]
  15. Error fetching PMID 7966320: [Wilson94]
  16. Error fetching PMID 7568019: [Wilson95]
  17. Error fetching PMID 1703438: [Yager91]
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