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Transcriptional Regulation Getting started Promotors, Sigma Factors, and DNA-binding proteins

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Promotors -10 and -35 consensus sequences (before transcription, not start codon) -10 TATAAT TATA or Pribnow Box -35 TTGACA T-T-GA-CA Altered sequence weak promotor Sequence complementary to sigma factor of RNA pol

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Sigma factors vary First example of global regulation simultaneous, coordinated control of multiple genes and operons Table 8.2 Brock 11th

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DNA-binding Proteins Sigma factors 70 Activators Repressors

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Helix-turn-Helix Motif

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Features of the Interaction Repressors often act as dimers or tetramers Each monomer has recognition domain and stabilization domain Recognition sequence often involves inverted repeats figure 8.8 Brock 11th

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Eukaryotes feature Zinc-fingers and Leucine zippers (figure 8.10)

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Transcriptional Regulation Lets be positive

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Positive regulators - activators Activator binds to activator site or enhancer site upstream of promotor Facilitate RNA pol binding to promotor Actual touching RNA pol Melting

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Activator binding to DNA may require small molecule - inducer Examples AraC protein binds L- arabinose, and then the L-ara promotor Maltose and the mal operon (figure 8.15) cAMP + cAMP Receptor Protein (CRP) will be considered in detail later

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Enhancer sites or activator-binding sites can occur distant from the promotor Results in bending of DNA May result in opening of promotor double helix Bent DNA may be required for RNA pol-activator complex to form Example NR I -P activator of the ntr regulon May involve Integration Host Factor (IHF) protein

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Transcriptional Regulation Negative control of transcription: Repression and induction

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Importance of operator region Protein binds operator and blocks RNA pol LexA repressor protein blocks synthesis of DNA repair enzymes like uvrABC When DNA is damaged, RecA protein becomes a protease that specifically degrades LexA protein

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Repressor frequently interacts with small molecule (effector) Presence of small molecule prevents transcription Frequently involved in control of amino acid synthesis (anabolic) genes Prevents costly synthesis of unnecessary proteins

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Repression involves corepressor molecule binding to aporepressor protein arginine (corepressor) binds (apo-)repressor that binds operator (fig. 8.13)

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One level of control of tryptophan biosynthesis TrpR protein 11 kD, acts as a dimer, 50 copies per cell Binds operator when tryptophan is present Autogenous regulation also will block its own synthesis

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Small molecules (inducers) can bind repressor protein and prevent binding to operator Enzymes will be synthesized only when inducer is present Typically involves catabolic enzymes Utilization of particular sugars

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lac Operon Simple Version Inducer binds repressor protein and reduces affinity for operator Actual inducer is allolactose (an isomer of lactose) Artificial inducer is isopropyl--D- thiogalactoside lac operon only transcribed if lactose is available

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Transcriptional Regulation Reduction of transcription after initiation: Attenuation of the trp operon

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Key Features of Attenuation Leader region (trpL) occurs between promotor and first gene (trpE) Leader region peptide requires 2 charged trp-tRNA Inverted repeats lead to stem loop structures (including a terminator) A second ribosome is needed (this is the secret nobody talks about)

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trp mRNA Synthesis at Low [tryptophan] 10% of full expression RNA pol slides along DNA, making transcript Ribosome starts translating message Ribosome sails through region 1 containing tryptophan codons Ribosome reaches stop codon and falls off

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trp mRNA Synthesis at Low [tryptophan] 10% of full expression Consequences Leader peptide is completed Region 1 is free to pair with region 2 Region 3 is free to pair with region 4 3:4 Stem loop is a termination stem loop and RNA pol falls off no mRNA!

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Lets take a closer look

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Lets take an even closer look Shine- Delgarno

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trp mRNA Synthesis at Very Low [tryptophan] full expression RNA pol slides along DNA, making transcript Ribosome starts translating message Ribosome stalls at tryptophan codon

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trp mRNA Synthesis at Very Low [tryptophan] full expression Consequences Leader peptide is not completed Region 1 cant pair with region 2 Region 2 is free to pair with region 3 3:4 termination stem loop does not form and RNA pol continues to trpE

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Lets take a closer look - again Ribosome stalls here

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Lets take an even closer look Shine- Delgarno

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Attenuation is a widespread control mechanism for amino acid synthesis Threonine Phenylalanine Histidine 7 straight His! No operator needed all attenuation control