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Department
Biochemistry
Course
Biochemistry 2280A
Professor
Eric Ball
Semester
Fall

Description
Lecture 2 – Brandl Regulation of Gene Expression: - How are different bacterial promoters transcribed at different levels? There are 2 key reasons why some bacterial promoters are transcribed more than others: 1. Strength of the basic promoter elements – not all -10 and -35 sequences are equally active  They bind RNA polymerase holoenzyme with different affinities  2. More than one sigma factor  Each recognizes different promoters  The most prevalent is the “housekeeping” sigma, 70 54 HS  Others include… (nitrogen metabolism) and  (heat shock) 3. Gene specific regulatory proteins bind specific DNA sequences found in one or more promoters and serve to activate or repress transcription  Dynamic!  Positive regulation – factors activate transcription – an activator protein (CAP) regulates transcription of the lac operon and other genes involved in carbon metabolism  Negative regulation – factors repress transcription - tryptophan (Trp) repressor Trp Operon:  In E. coli, the 5 genes (E, D, C, B, A) for tryptophan biosynthesis are transcribed as a single unit from a common promoter  The Trp operon is expressed when there is a little tryptophan in the cellular environment  When tryptophan concentration is low… o Tryptophan dissociates from the Trp promoter – Trp repressor no longer binds the Trp operator o RNA polymerase can access the Trp promoter – initiates transcription o RNA polymerase is recruited to the Trp promoter by the -10 and -35 sequences allowing transcription o Cell wants to turn on gene biosynthesis when little Trp around – if it cant use Trp from environment, it will make its own gene o At low concentrations of tryptophan, the tryptophan comes off and now there is a structural change in the protein - the alhpa-5 helices tilt inwards and this form of the protein can no longer make good contacts to the DNA  When tryptophan concentration is high… o The Trp repressor binds tryptophan operon and blocks expression o A conformational change occurs in the repressor’s structure such that it can bind the operator site within the promoter o Binding of the repressor sterically inhibits access of RNA polymerase to the promoter Trp promoter:  Between the -35 and -10 sequences is an element called the Trp operator that binds a protein called the Trp repressor  At high concentrations of tryptophan, Trp repressor binds the amino acid tryptophan What is the structural basis for this regulation?  Trp repressor – monomer o 107 amino acid residues o Has a helix turn helix motif that is required for DNA binding o Alpha helices 4 and 5 make up the helix turn helix motif – make base specific interaction with DNA o Binds DNA as a dimer  Trp repressor – dimer o Protein dimer has 2 fold symmetry o Amino acid side chains on helix 5 make base specific contacts with the major groove of its operator sequence The Trp repressor dimer with the core of the protein held together via protein-protein interactions  (a) Trp repressor in the absence of bound tryptophan – it cannot bind the operator DNA since the recognition helices are tilted inward  (b) Active Trp repressor (capable of binding its operator) when tryptophan is bound – recognition helix 5 on each of the 2 monomers makes base specific contacts in consecutive major grooves of the DNA General themes to take from the Trp operon: 1. Trp repressor is a site specific DNA binding protein o Protein makes base specific contact based on the DNA sequence o Only want binding in certain spots of the genome 2. There is a binding site for Trp repressor within the Trp promoter 3. Trp repressor inhibits transcription by blocking access of RNAP to the promoter 4. Trp repressor is responsive to an environmental signal o In this case, it is responsive to tryptophan o Can turn genes on and off in response to tryptophan Positive Control:  Gene specific regulatory proteins can also act to enhance transcription  Increased transcription is generally accomplished by increasing the rate of recruitment of RNA polymerase or the activity of RNA polymerase  Both enhanced recruitment and/ or enhanced activity of RNA polymerase are usually achieved by protein-protein interactions between the activator protein and RNA polymerase Lac Operon of E.coli:  The Lac operon contains those genes required for the metabolism of lactose in E.coli  Expression of lac is controlled by 2 signals… o First is negative regulation similar to what is seen for the Trp operon o Second mechanism for regulating the lac operon involves control by glucose  Operon is turned on when lactose is around o Not required for biosynthesis of lactose, but is used to metabolize lactose and get energy from it Negative Regulation at the Lac Operon:  Cells only require expression of the lac operon if lactose is in the media  In the absence of lactose… o The lac repressor binds the promoter, inhibiting the action of RNA polymerase o Inhibits transcription  In the presence of lactose… o The lac repressor binds lactose - results in a conformational change in the protein so that it no longer binds the operator sequence in the promoter (dissociates from the operator) o Thus allowing RNA polymerase to come in and transcription to occur  -10 and 35 are not very strong here *Positive
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