Gene regulation

Positive vs. negative control Positive control: A protein binds to DNA and transcription increases mRNA 5’ 3’ CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -35 -10 +1 Activator Negative control: A protein binds to DNA and transcription decreases X mRNA 5’ 3’ CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -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 X mRNA LacI 5’ 3’ CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -35 -10 +1 DNA covered by repressor Operator Without lactose, LacI binds to the operator, which prevents RNA polymerase from binding to the promoter

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

The lac operon: negative regulation Promoter DNA covered by RNA polymerase mRNA 5’ 3’ CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -35 -10 +1 DNA covered by repressor LacI Operator When LacI 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 LacI to the operator with and without lactose. Which curve has a higher Keq for binding to DNA? 100 % DNA bound 50 [LacI] The red curve Keq Operator DNA + LacI LacI/Operator Complex [O] [R] [O-R] [O−R] O [R] 1 R Keq for binding is inversely related to the concentration of LacI at which 50% of the DNA is bound Keq = =

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

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

The lac operon: positive regulation CAP, the activator, can only bind to the DNA when cAMP levels are high. This only occurs when glucose levels are low. CAP Binding Site Promoter mRNA CAP 5’ 3’ CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -35 -10 +1 Operator CAP + cAMP

The lac operon: positive regulation CAP, the activator, can only bind to the DNA when cAMP levels are high. cAMP levels are high when glucose levels are low. CAP Binding Site Promoter mRNA CAP 5’ 3’ CAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACATTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCT GTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTCCGAAATGTAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA | | | -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? [repressor] 10-12 10-11 10-10 10-9 10-8 [DNA] Constant without molecule A Without molecule A: Keq = 109 With molecule A: Keq = 1011 [repressor] 10-12 10-11 10-10 10-9 10-8 [DNA] Constant 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. with molecule A

The lac operon Glucose CAP-Binding Site -10 Region LacZ RNA Polymerase LacI cAMP Consensus Sequence Lactose Repressor CAP Activator Promoter Operator

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