Artificial transcriptional terminators

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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]
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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]
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*side note
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*Effects of stem loop sequence on terminator efficiency
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Several sources [Reynolds 92, Chamberlin 79] measured the termination effiecency(TE) of T7Te at around 90%.  However, effiency for the biobricks part BBa_B0012 [http://parts.mit.edu/registry/index.php/Part:BBa_B0012], also T7Te, is around 30%T7Te has a very short poly(U) tail and requires the further downstream sequence for effiecent 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.
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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.
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*T7Te
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Several sources [Reynolds 92, Chamberlin 79] measured the termination efficiency(TE) of T7Te at around 90%.  However, efficiency for the biobricks part BBa_B0012 [http://parts.mit.edu/registry/index.php/Part:BBa_B0012], 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.
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*Predicting terminator efficiency
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It may be possible to predict terminator efficiency using methods from d'Aubenton, in particular, the score d assigned to a possible terminator sequence
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<math>''d'' = nt*18.16+Y*96.59-116.87</math>
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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.
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The score d will give a rough estimate of how efficient a terminator is.
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d<0:  TE<20%
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0<d<30:  20%<TE<70%
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d>30:  TE>70%
===References===
===References===
<biblio>
<biblio>

Revision as of 17:00, 17 August 2006

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

  1. Abe H and Aiba H. . pmid:9150882. PubMed HubMed [Abe96]
  2. d'Aubenton Carafa Y, Brody E, and Thermes C. . pmid:1702475. PubMed HubMed [Aubenton90]
  3. Brendel V, Hamm GH, and Trifonov EN. . pmid:3078109. PubMed HubMed [Bredel86]
  4. Cheng SW, Lynch EC, Leason KR, Court DL, Shapiro BA, and Friedman DI. . pmid:1835546. PubMed HubMed [Cheng91]
  5. Christie GE, Farnham PJ, and Platt T. . pmid:7027254. PubMed HubMed [Christie81]
  6. Ermolaeva MD, Khalak HG, White O, Smith HO, and Salzberg SL. . pmid:10926490. PubMed HubMed [Ermolaeva00]
  7. Lesnik EA, Sampath R, Levene HB, Henderson TJ, McNeil JA, and Ecker DJ. . pmid:11522828. PubMed HubMed [Lesnik01]
  8. Lynn SP, Kasper LM, and Gardner JF. . pmid:2961747. PubMed HubMed [Lynn88]
  9. Petrillo M, Silvestro G, Di Nocera PP, Boccia A, and Paolella G. . pmid:16820051. PubMed HubMed [Petrillo06]
  10. Reynolds R, Bermúdez-Cruz RM, and Chamberlin MJ. . pmid:1372365. PubMed HubMed [Reynolds92I]
  11. Reynolds R and Chamberlin MJ. . pmid:1372366. PubMed HubMed [Reynolds92II]
  12. Unniraman S, Prakash R, and Nagaraja V. . pmid:11809879. PubMed HubMed [Unniraman02]
  13. Uptain SM and Chamberlin MJ. . pmid:9391063. PubMed HubMed [Uptain97]
  14. von Hippel PH and Yager TD. . pmid:1536005. PubMed HubMed [VonHippel92]
  15. Wilson KS and von Hippel PH. . pmid:7966320. PubMed HubMed [Wilson94]
  16. Wilson KS and von Hippel PH. . pmid:7568019. PubMed HubMed [Wilson95]
  17. Yager TD and von Hippel PH. . pmid:1703438. PubMed HubMed [Yager91]
All Medline abstracts: PubMed HubMed
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