Talk:CH391L/S12/TranscriptionPromotersandTerminators

• Midhat Patel 15:50, 27 February 2012 (EST): Can sigma factors be used to regulate gene expression? If we use a promoter associated with a sigma factor that operates under specific conditions, can it be used as a promoter for a system and integrated into E. coli?
  • Yi Kou 10:07, 3 March 2012 (EST): Good idea! Here is one paper of similar "genetic transplantation" of sigma factor[1]. But I think for actual application, it is not so simple as one factor and binding sequence could do the work. Many regulations, involving posttranslational regulation and auto-regulation have been confirmed for many types of sigma factors. And if these accompanies are not well considered, I think it would be hard to observe the expected result or phenotype. Also, it has been suggested that the sigma factor is not "on and off" the core pol but rather shows a "self-changing" pattern associated with the RNA pol[2]. This might add another influence of "interaction spectrum" with this sigma factor "transplantation" idea.
    • James L. Bachman 15:33, 4 March 2012 (EST): Going off of what Yi said, I think that regulating gene expression with a sigma factor would not be very practical because it would be difficult to implement due to the many factors involved. If you wanted to transcribe something under specific conditions, it would be much easier to use an inducible promoter and selectively add the exogenous agent to force transcription under the condition you want. Gourse et al. showed that sometimes multiple sigma factors could use the same promoter if the sigma factors recognized similar sequence consensus sequences.[3] If you wanted to promote transcription with only one sigma factor, then it might be possible to do as they did and inactivate the other sigma factors that could use that promoter.

• Jeffrey E. Barrick 11:16, 3 March 2012 (EST):How did they create the "insulator" sequence that didn't have any known transcription factor binding sites? Did they make it up from scratch, or did they grab it from somewhere else in a different genome?
  • Yi Kou 11:49, 3 March 2012 (EST):for the insulator, one way to detect [4].
    • James L. Bachman 15:33, 4 March 2012 (EST): The most detail I could find the article about the design of the insulation was that they "generated promoters in which adjoining upstream and downstream sequences, which potentially could alter transcription initiation and promoter escape, were included in the promoter cassette."[5] The author doesn't explicitly say it was taken from another genome, and the methods and material only talks about how the promoter was inserted into plasmid. It seems like they developed the insulation from scratch, although I can't say for certain.

• Ben Slater 15:47, 3 March 2012 (EST): In your section on promoters, you say "there has not been a promoter found in E. coli that is of the consensus sequence, it would likely bind so strongly that elongation would not occur." Since transcription rate increases as the promoter approaches the consensus sequence, but plummets if it actually IS the consensus sequence, where is the cutoff? Will only 1 bp difference be the optimal rate?
  • James L. Bachman 15:33, 4 March 2012 (EST): It is true that as the promoter sequence gets closer to the consensus sequence the strength of the promoter increases. However, the similarity of a promoter at -10 and -35 to the consensus sequence does not necessarily mean the promoter will automatically be strong. [6] From this it can be seen that generally those promoters that are similar to consensus seem much stronger, but it is also dependent on the 17 bp spacer in between the sequences the length from the -10bp sequence to the transcription start site. The spacing is necessary for the sigma factors of the RNAP holoenzyme to bind properly. As well, the sequence between the -10 and the start site is necessary to be 7 bp because the promoter has to escape or it cannot elongate. Assuming that these factors are constant, then I think that you are correct in that a promoter that differs in only 1 bp from the consensus would be the strongest promoter. [7]

References

1. Karlinsey JE and Hughes KT. Genetic transplantation: Salmonella enterica serovar Typhimurium as a host to study sigma factor and anti-sigma factor interactions in genetically intractable systems. J Bacteriol. 2006 Jan;188(1):103-14. DOI:10.1128/JB.188.1.103-114.2006 |
2. Mukhopadhyay J, Kapanidis AN, Mekler V, Kortkhonjia E, Ebright YW, and Ebright RH. Translocation of sigma(70) with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA. Cell. 2001 Aug 24;106(4):453-63. DOI:10.1016/s0092-8674(01)00464-0 |
3. Newlands JT, Gaal T, Mecsas J, and Gourse RL. Transcription of the Escherichia coli rrnB P1 promoter by the heat shock RNA polymerase (E sigma 32) in vitro. J Bacteriol. 1993 Feb;175(3):661-8. DOI:10.1128/jb.175.3.661-668.1993 |
4. Burgess-Beusse B, Farrell C, Gaszner M, Litt M, Mutskov V, Recillas-Targa F, Simpson M, West A, and Felsenfeld G. The insulation of genes from external enhancers and silencing chromatin. Proc Natl Acad Sci U S A. 2002 Dec 10;99 Suppl 4(Suppl 4):16433-7. DOI:10.1073/pnas.162342499 |
5. Davis JH, Rubin AJ, and Sauer RT. Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Res. 2011 Feb;39(3):1131-41. DOI:10.1093/nar/gkq810 |

6. http://www.nature.com/msb/journal/v3/n1/extref/msb4100187-s3.pdf

7. Cox RS 3rd, Surette MG, and Elowitz MB. Programming gene expression with combinatorial promoters. Mol Syst Biol. 2007;3:145. DOI:10.1038/msb4100187 |