The attenuation mechanism for regulating the trp operon of E. coli may have components not described in your textbook
|Main area:||Molecular biology|
|Target group:||Biochemistry, Biology, Molecular Biomedicine|
|Educational level:||Masters, Bachelor|
This regulatory mechanism for governing the synthesis of the tryptophan biosynthesis enzymes is described in most Molecular Biology textbooks. Briefly, the early part of the mRNA for the trp enzymes can form two alternative structures – one that terminate transcription and one that allow the RNA-polymerase to transcribe into the structural trp genes.
Which of the two structures that is formed is determined by the translation rate for two adjacent trp codons in a 14 codon long leader-gene early on the mRNA. If the translation is slow due to lack of tryptophan, the non-terminated mRNA that reads into the trp genes is made. Fast translation of the leader peptide signals plenty of tryptophan in the cell and the alternative RNA-polymerase terminating structure is then formed and the cell stop making the trp mRNA and trp enzymes.
Very elegant, but we know now that in bacteria there should be a physical contact, mediated by the NusG protein, between the RNA-polymerase and the ribosomal protein S10 in the initial Ribosome that is translating the mRNA. If this contact is abscent, the RNA-polymerase will be terminated by the transcription termination factor Rho. It is therefore difficult to envisage how the RNA-polymerase could get away from the initial Ribosome and make the mRNA that then could fold differently.
The newly formed mRNA might "spool out" and form the structures, but alternatively the attenuation mechanism might only work in the time window between when the sigma subunit has left the polymerase after initiation and before the NusG protein has bound to the RNA-polymerase.
We have initial supporting evidence for this model, but want further evidence.
The project I propose, will investigate what would happen to the attenuation mechanism if one manipulated the duration of this time window.
We have a plasmid carrying an inducible version of the NusG protein. First a trp-lacZ fusion should be obtained or made, and then the rate of synthesis of the lacZ enzyme should be measured with and without excess NusG protein. Excess NusG should diminish this time window.
Direct measurements of RNA polymerase termination should also be done. This could for example be done by a length determination of the mRNA by Northern blotting.
Finally, one might predict that attenuation is only possible if the leader peptide is located very early on the mRNA, because the time window would disappear with time. This could be tested by inserting an additional gene between the trp-promoter and the gene for the leader peptide. When the polymerase in this construct reach the gene for the leader peptide the NusG protein would have bound the RNA-polymerase and the attenuation mechanicm should not function according to this prediction. This Master project could be divided into smaller projects for Bachelor students.
If you are interested and want to hear more please contact Steen Pedersen at: firstname.lastname@example.org
|Methods used:||Enzyme assays, Northern Blot, possibly Mathematical modeling|
|Keywords:||cloning of genes, protein expression|