lot confused; mechanism of bacterial gene expression ?

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rucshaca
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lot confused; mechanism of bacterial gene expression ?

Post by rucshaca » Thu Oct 27, 2005 2:16 am

Hi All,

I am in a big confused state about a question been asked to me in my college. I would really really appreciate if someone can please help me in giving some directions or throw some light in answering to this.

I do not know what is expected from the originator of this question and it is been passed on to me as a challenge. All i know is that the question is tricky and answer may be simple ( :-) )



Here is what has been asked:

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The regulation of bacterial genes occurs via one of the three general mechanisms.

1) through regulated recruitment of polymerase

2) through regulated polymerase activation

3) through regulated promoter activation

Each involves an activator protein (+/ - a separate sensor protein) which works on its target in a different manner.

You have been asked to look at the regulatory circuit controlling the expression of a bacterial gene X through an activator protein Y. Assume you have cloned each of these genes (including the separate sensor if necessary), know the promoter as well as all other relevant binding sites etc. and have access to any legitimate experimental techniques in molecular biology/genetics.


Outline/Design a set of experiments that will tell you the overall mechanism (1,2 or 3) by which the activator works. Mention the expected outcome of each experiment if the mechanism were 1 or 2 or 3 and explain why this would be so.

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PLEASE ADVISE AS YOUR RESPONSE WILL BE VERY FRUITFUL.

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GreenDog
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Post by GreenDog » Thu Oct 27, 2005 8:55 am

Just my thoughts on the subject... which might not have any connection with reality...
You construct a system where you have: Gene X with endogenous promoter Gene Y under inducible prompter, Gene Z endogenous unrelated prompter which is not supposed to be regulated by Y, for control.
Make sure that; Z is expressed regardless of Y expression, when Y is induced X is expressed, when Y is not induced X is not expressed.
Now, stop the production of Y.
1. If when we don't have Y the polymerase is not attached (some binding assay), and when we have Y it is attached, it's possibility 1.
2.If Y copersipitates with the polymerase it's 2.
3.If Y binds DNA it's 3.
You can assume the mechanisms are mutually exclusive or do all the experiments.

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