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Brandl Lecture 2.docx

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Biochemistry 2280A
Chris Brandl

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Brandl Lecture 2 – Regulation of Gene Expression How are different bacterial promoters transcribed at different levels? - Some sequences have better -10 and -35 sequences  better attraction than others o Will be lower if they cannot attract as well o Not dynamic – cell cannot change -10 and -35 sequences if it wants to up or down regulate that amount of expression - There is more than one sigma factor  each recognizes different promoters o Different sigma factors recognize different sequences o The promoter shown in lecture 1 was for house-keeping sigma/sigma 70 o Most prevalent is housekeeping sigma o Others include sigma 54 in nitrogen metabolism that will turn on metabolism, sigma HS for heat shock – when heat shocked, becomes activated and then turns on in the presence of high temperature  Found in bacteria but not in eukaryotes – conserved for bacteria - Gene specific regulatory proteins – DYNAMIC o Negative regulation – factors repress transcription – trp operon and trp repressor o Positive regulation – factors activate transcription – eukaryotic reduction system Negative regulation – factors inhibiting Trp operon – in E. coli the 5 genes (E, D, B, C, A) for tryptophan biosynthesis are transcribed from a common promoter - Five proteins are required for tryptophan biosynthesis – enzymes are made from this - Trp operon is regulated by concentration of trp in the environment/medium o Trp production is induced when there is little trp in the environment - Q: When is trp operon expressed? A: When there is little tryptophan in cellular environment How is the trp operon regulated by tryptophan? - Trp promoter – typical with +1 start site and -35 and -10 - Special sequence between -35 and -10 sequences called trp operator that binds a protein called the trp repressor o Not true for every promoter but true for trp operon o At high *trp+, trp repressor binds trp o The trp repressor-trp complex binds the operator DNA What happens to transcription? - The trp repressor-trp complex blocks RNA polymerase from the promoter so that no transcription is possible  allosterically inhibits transcription - When *trp+ is low, trp dissociates from trp repressor - The trp repressor no longer binds to the operator - RNA polymerase can access trp promoter. Transcription occurs - When genes are off = high *trp+; when genes are on = low *trp+ What is the structural basis for regulation by trp? - Trp repressor – monomer that does not have catalytic activity o 107 amino acid residues, mostly alpha helices o Alpha helices 4 and 5 make up helix-turn-helix motif that binds the DNA o Works as a dimer o Protein dimer has 2 fold symmetry o 2 alpha Helix 3s contact to allow dimerization o Many DNA binding proteins act as dimers or pseudo-dimers o Helix 5 of each monomer recognizes adjacent major grooves in the operator DNA  Perfect size to fit into major groove  Most base-specific contact made with major groove since minor groove is too small to fit alpha helix - Tryptophan binding induces a conformational change in the trp repressors which allows DNA binding o Alpha helix 5 tilts inwards when trp is low and cannot interact with DNA o Affinity for DNA drops dramatically when protein is not bound by trp o Molecular basis – causes conformational change allowing for binding of DNA - General themes to take from trp operon o Trp repressor is site specific DNA binding protein – operator sequence between -10 and - 35  regulates set of genes that you want regulator to bind to set of genes o There is a binding site for trp repressor within trp promoter o Trp repressor inhibits transcription by blocking access of RNAP to the promoter  Repression works in similar ways by stopping RNAP from getting to promoter o Trp repressor is responsive to environmental signal  Allows it to be turned on and off so it must recognize some signal  Trp specifically represses trp repressor Q: Genetics has been critical in understanding the regulation of the trp operon. Different mutations in the trp repressor can result in? A: the trp operon always being transcribed and the trp operon never being transcribed - Mutations in trp repressor the prevent binding to operator – trp operon always being transcribed - Mutations in the trp repressor that allow it to bind the operator in the absence of trp – trp operon never being transcribed Six differences in transcription between prokaryotes and eukaryotes 1. Three DNA polymerases in eukaryotes but 1 in prokaryotes a. Pol 1 – most rRNA genes b. Pol 2 – mRNA’s c. Pol 3 – tRNAs, 5S rRNA 2. No operons in eukaryotes a. Each gene is transcribed as a single unit i. Monocistronic (one RNA, one protein) – in contrast to polycistronic in bacteria (one protein, multiple RNA) 3. Promoter structure a. Eukaryotes do not have -10 and -35 sequences and no sigma factor b. Promoter recognition is determined by a set of proteins, one of which recognizes a TATA element - TATA box in gene that looks a little like -10 and recruits factors TF2D which has TATA binding
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