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Regulation of Gene Expression

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Biochemistry 2280A
Derek Mc Lachlin

Brandl Lecture 2 Notes: REGULATION OF GENE EXPRESSION 11/09/2012 Methods of Regulation 1. Some genes have better -10 and -35 sequences  These genes will be expressed at higher levels  W ill recruit RNA polymerase at higher efficiency  Not dynamic  Sequences cannot be regulated; are “stuck” with that sequences throughout time 2. There is more than one sigma factor  Each recognizes different promoters  The most prevalent is the “housekeeping” σ, σ70 54 HS  Others include σ nitrogen metabolism, σ heat shock  Heat shock responds to high temperature, is induced and reacts with RNA core enzyme 3. Gene specific regulatory proteins  Dynamic!  Can change genes in response to different environmental signals through these gene specific regulatory proteins  Largely used by eukaryotic cells  Negative regulation – factors repress transcription  Positive regulation – factors activate transcription Negative Regulation Example: trp operon  In E. coli the 5 genes for tryptophan biosynthesis are transcribed from a common promoter  The trp operon is regulated by the concentration of tryptophan in the environment  Trp operon is expressed when there is little tryptophan in the cellular environment  How is the trp operon regulated by tryptophan? Trp Promoter  σ regulated promoter  Between the -10 and -35 sequences is an element called the trp operator that binds a protein called the trp repressor  At high [tryptophan], trp repressor binds tryptophan  The trp repressor-tryptophan complex binds the operator DNA  The trp-repressor-tryptophan complex blocks RNA polymerase from the promoter  RNA polymerase cannot bind to the promoter; it is sterically inhibited from getting there  Polymerase can’t park at the promoter  When [tryptophan] is low, tryptophan dissociates from the trp repressor  The trp repressor no longer binds the trp operator  The trp repressor will only bind DNA when it itself is bound to tryptophan Result  RNA polymerase can access the trp operator, transcription occurs  Low [tryptophan] – genes are on  High [tryptophan] – genes are off  What is the structural basis for the regulation by tryptophan? Trp Repressor – Monomer  107 amino acid residues  Alpha helices 4 and 5 make up the helix-turn-helix motif  Helices 4 and 5 are critical  Helix-turn-helix motif is crucial for binding DNA  Many DNA-binding proteins act as dimers  Can be true dimers like (trp repressor) or pseudo-dimers Trp Repressor – Dimer  Protein dimer has 2-fold symmetry  Helix 3s contact to allow dimerization  Helix 5 of each monomer recognizes adjacent major grooves in the operator DNA  Major groove is wide enough to fit an alpha helix  Protein can read the bases in the major groove  Most proteins interact with major groove, not minor groove  Minor groove is too small, doesn’t have much discriminatory power  Repressor only binds to DNA when it has tryptophan bound to it  Tryptophan binding induces a conformational change in the Trp repressor which allows DNA binding  Binding of tryptophan is crucial to remaining conformation of protein that will bind to DNA  Tryptophan binds to protein, alters conformation of protein (from tilted helix 5s to new conformation)  Process has been selected for through evolution General Themes to take from the Trp Operon 1. Trp repressor is a site specific DNA binding protein  Binding to specific sequences very tightly  Recognize individual specific sequences 2. There is a binding site for trp repressor within the Trp promoter  Genes that don’t have an operator won’t be regulated by the trp repressor; binding site is needed 3. Trp repressor inhibits transcription by blocking access of RNAP to the promoter 4. Trp repressor is responsive to an environmental signal  For Trp repressor, this would be tryptophan (environmental signal)  All gene specific regulatory proteins are regulated by some signal  Q: Genetics has been critical in understanding the regulation of the trp operon. Dif
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