Difference between revisions of "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]
  
*side note
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*Effects of stem loop sequence on terminator efficiency
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.
  
 +
*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>
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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 14:00, 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

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. Differential contributions of two elements of rho-independent terminator to transcription termination and mRNA stabilization. Biochimie. 1996;78(11-12):1035-42. PubMed ID:9150882 | HubMed [Abe96]
  2. d'Aubenton Carafa Y, Brody E, and Thermes C. Prediction of rho-independent Escherichia coli transcription terminators. A statistical analysis of their RNA stem-loop structures. J Mol Biol. 1990 Dec 20;216(4):835-58. PubMed ID:1702475 | HubMed [Aubenton90]
  3. Brendel V, Hamm GH, and Trifonov EN. Terminators of transcription with RNA polymerase from Escherichia coli: what they look like and how to find them. J Biomol Struct Dyn. 1986 Feb;3(4):705-23. DOI:10.1080/07391102.1986.10508457 | PubMed ID:3078109 | HubMed [Bredel86]
  4. Cheng SW, Lynch EC, Leason KR, Court DL, Shapiro BA, and Friedman DI. Functional importance of sequence in the stem-loop of a transcription terminator. Science. 1991 Nov 22;254(5035):1205-7. PubMed ID:1835546 | HubMed [Cheng91]
  5. Christie GE, Farnham PJ, and Platt T. Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4180-4. PubMed ID:7027254 | HubMed [Christie81]
  6. Ermolaeva MD, Khalak HG, White O, Smith HO, and Salzberg SL. Prediction of transcription terminators in bacterial genomes. J Mol Biol. 2000 Aug 4;301(1):27-33. DOI:10.1006/jmbi.2000.3836 | PubMed ID:10926490 | HubMed [Ermolaeva00]
  7. Lesnik EA, Sampath R, Levene HB, Henderson TJ, McNeil JA, and Ecker DJ. Prediction of rho-independent transcriptional terminators in Escherichia coli. Nucleic Acids Res. 2001 Sep 1;29(17):3583-94. PubMed ID:11522828 | HubMed [Lesnik01]
  8. Lynn SP, Kasper LM, and Gardner JF. Contributions of RNA secondary structure and length of the thymidine tract to transcription termination at the thr operon attenuator. J Biol Chem. 1988 Jan 5;263(1):472-9. PubMed ID:2961747 | HubMed [Lynn88]
  9. Petrillo M, Silvestro G, Di Nocera PP, Boccia A, and Paolella G. Stem-loop structures in prokaryotic genomes. BMC Genomics. 2006 Jul 4;7:170. DOI:10.1186/1471-2164-7-170 | PubMed ID:16820051 | HubMed [Petrillo06]
  10. Reynolds R, Bermúdez-Cruz RM, and Chamberlin MJ. Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators. J Mol Biol. 1992 Mar 5;224(1):31-51. PubMed ID:1372365 | HubMed [Reynolds92I]
  11. Reynolds R and Chamberlin MJ. Parameters affecting transcription termination by Escherichia coli RNA. II. Construction and analysis of hybrid terminators. J Mol Biol. 1992 Mar 5;224(1):53-63. PubMed ID:1372366 | HubMed [Reynolds92II]
  12. Unniraman S, Prakash R, and Nagaraja V. Conserved economics of transcription termination in eubacteria. Nucleic Acids Res. 2002 Feb 1;30(3):675-84. PubMed ID:11809879 | HubMed [Unniraman02]
  13. Uptain SM and Chamberlin MJ. Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates. Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13548-53. PubMed ID:9391063 | HubMed [Uptain97]
  14. von Hippel PH and Yager TD. The elongation-termination decision in transcription. Science. 1992 Feb 14;255(5046):809-12. PubMed ID:1536005 | HubMed [VonHippel92]
  15. Wilson KS and von Hippel PH. Stability of Escherichia coli transcription complexes near an intrinsic terminator. J Mol Biol. 1994 Nov 18;244(1):36-51. DOI:10.1006/jmbi.1994.1702 | PubMed ID:7966320 | HubMed [Wilson94]
  16. Wilson KS and von Hippel PH. Transcription termination at intrinsic terminators: the role of the RNA hairpin. Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8793-7. PubMed ID:7568019 | HubMed [Wilson95]
  17. Yager TD and von Hippel PH. A thermodynamic analysis of RNA transcript elongation and termination in Escherichia coli. Biochemistry. 1991 Jan 29;30(4):1097-118. PubMed ID:1703438 | HubMed [Yager91]
All Medline abstracts: PubMed | HubMed