Gene regulation Positive vs negative control Positive control

Gene regulation

Positive vs. negative control Positive control: A protein binds to DNA and transcription increases m. RNA 5’ 3’ CAACGCAATTAATGTGAGTTAGCTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -35 -10 +1 Activator Negative control: A protein binds to DNA and transcription decreases 5’ 3’ X m. RNA CAACGCAATTAATGTGAGTTAGCTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -35 -10 +1 Repressor

The lac operon What is an operon? A collection of genes that are transcribed together and often have a related function (in this case lactose metabolism). Lactose

The lac operon: negative regulation Promoter DNA covered by RNA polymerase 5’ 3’ Lac. I X m. RNA CAACGCAATTAATGTGAGTTAGCTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -35 -10 +1 DNA covered by repressor Operator Without lactose, Lac. I binds to the operator, which prevents RNA polymerase from binding to the promoter

The lac operon: negative regulation Promoter DNA covered by RNA polymerase 5’ 3’ Lac. I X m. RNA CAACGCAATTAATGTGAGTTAGCTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -35 -10 +1 DNA covered by repressor Lac. I Operator + Lactose If lactose is added, it binds Lac. I and stabilizes the Lac. I conformation that doesn’t bind to the operator.

The lac operon: negative regulation Promoter DNA covered by RNA polymerase m. RNA 5’ 3’ CAACGCAATTAATGTGAGTTAGCTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -35 -10 +1 DNA covered by repressor Lac. I Operator When Lac. I is not bound to the operator, the promoter binding site is not occluded.

Keq of DNA binding The graph to the right shows two curves that represent binding of Lac. I to the operator with and without lactose. 100 % DNA bound Which curve has a higher Keq for binding to DNA? 50 0 [Lac. I] The red curve Operator DNA + Lac. I [O] Keq = = [R] Keq Lac. I/Operator Complex [O-R] Keq for binding is inversely related to the concentration of Lac. I at which 50% of the DNA is bound

Keq of DNA binding The graph to the right shows two curves that represent binding of Lac. I to the operator with and without lactose. Which curve represents binding of Lac. I without lactose? Higher Keq 100 % DNA bound Lower Keq 50 0 [Lac. I] The red curve – Lac. I has a higher affinity for DNA when lactose is absent.

The lac operon: positive regulation Dissociation of Lac. I from the operator is not sufficient to activate transcription of the lac operon, it also requires positive regulation. CAP Binding Site Promoter m. RNA 5’ 3’ CAACGCAATTAATGTGAGTTAGCTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -35 -10 +1 Why is positive regulation necessary? The lac promoter -10 and -35 regions poorly match the consensus sequence, and RNA polymerase binds poorly to them. Operator

The lac operon: positive regulation CAP, the activator, can only bind to the DNA when c. AMP levels are high. This only occurs when glucose levels are low. CAP Binding Site 5’ 3’ Promoter m. RNA CAP CAACGCAATTAATGTGAGTTAGCTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -35 -10 +1 Operator CAP + c. AMP

The lac operon: positive regulation CAP, the activator, can only bind to the DNA when c. AMP levels are high when glucose levels are low. CAP Binding Site 5’ 3’ Promoter m. RNA CAP CAACGCAATTAATGTGAGTTAGCTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -35 -10 +1 Operator CAP binding to the CAP binding site helps recruit RNA polymerase, allowing it to bind to the -35 and -10 sites that poorly match the consensus sequence.

Electrophoretic mobility shift assays (EMSA) The affinity of a certain repressor for binding to its operator site is affected by the presence of molecule “A. ” Given these EMSA data, would the addition of molecule A increase or decrease transcription of the gene controlled by this repressor? Without molecule A: Keq = 109 With molecule A: Keq = 1011 The affinity of the repressor for DNA is higher for higher when molecule A is present. The addition of molecule A would increase repressor binding to DNA, decreasing transcription. [repressor] 10 -12 10 -11 10 -10 10 -9 10 -8 [DNA] Constant without molecule A [repressor] 10 -12 10 -11 10 -10 10 -9 10 -8 [DNA] with molecule A Constant

The lac operon Glucose -10 Region RNA Polymerase Lac. I CAP-Binding Site Lac. Z Operon c. AMP -35 Region Consensus Sequence Lactose Repressor CAP Activator Promoter Operator

The lac operon: a concept map prevents Lac. I binding to the operator Lac. I binds to a site in the DNA called the CAP Binding Site lac Operon negatively regulates Operator positively regulates Promoter comprise CAP Binds to a site in the DNA called the Glucose encodes Lac. Z DNA that promotes transcription of is an Activator used to metabolize encodes the genes needed to use Is a Repressor Lactose binds -10 Region -35 Region RNA Polymerase binds Mediates CAP binding to DNA c. AMP is produced when glucose is depleted Consensus Sequence Ideal -10/-35 sequences to which RNA polymerase binds best