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] | + | 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 [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. | ||
| + | |||
| + | *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 | ||
| + | |||
| + | <math>''d'' = nt*18.16+Y*96.59-116.87</math> | ||
| + | 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=== | ===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
- Abe H and Aiba H. . pmid:9150882.
- d'Aubenton Carafa Y, Brody E, and Thermes C. . pmid:1702475.
- Brendel V, Hamm GH, and Trifonov EN. . pmid:3078109.
- Cheng SW, Lynch EC, Leason KR, Court DL, Shapiro BA, and Friedman DI. . pmid:1835546.
- Christie GE, Farnham PJ, and Platt T. . pmid:7027254.
- Ermolaeva MD, Khalak HG, White O, Smith HO, and Salzberg SL. . pmid:10926490.
- Lesnik EA, Sampath R, Levene HB, Henderson TJ, McNeil JA, and Ecker DJ. . pmid:11522828.
- Lynn SP, Kasper LM, and Gardner JF. . pmid:2961747.
- Petrillo M, Silvestro G, Di Nocera PP, Boccia A, and Paolella G. . pmid:16820051.
- Reynolds R, Bermúdez-Cruz RM, and Chamberlin MJ. . pmid:1372365.
- Reynolds R and Chamberlin MJ. . pmid:1372366.
- Unniraman S, Prakash R, and Nagaraja V. . pmid:11809879.
- Uptain SM and Chamberlin MJ. . pmid:9391063.
- von Hippel PH and Yager TD. . pmid:1536005.
- Wilson KS and von Hippel PH. . pmid:7966320.
- Wilson KS and von Hippel PH. . pmid:7568019.
- Yager TD and von Hippel PH. . pmid:1703438.
