The Operon 操縱元

1. The Operon 操縱元 a functioning unit of genomic material containing a cluster of genes under the control of a single regulatory signal or promoter

2. Ex Biochem c12-operon 12.1 Introduction Figure 12.1

3. Ex Biochem c12-operon 12.2 Regulation Can Be Negative or Positive • In negative regulation, a repressor protein binds to an operator to prevent a gene from being expressed. Figure 12.2

4. Ex Biochem c12-operon 12.2 Regulation Can Be Negative or Positive • In positive regulation, a transcription factor is required to bind at the promoter. • This enables RNA polymerase to initiate transcription. • Enhancer, activator Figure 12.3

5. Ex Biochem c12-operon 12.3 Structural Gene Clusters Are Coordinately Controlled • Genes coding for proteins that function in the same pathway may be: • located adjacent to one another • controlled as a single unit that is transcribed into a polycistronic mRNA Figure 12.4

6. Ex Biochem c12-operon 12.4 The lac Genes Are Controlled by a Repressor • Transcription of the lacZYA gene cluster is controlled by a repressor protein. • The repressor binds to an operator that overlaps the promoter at the start of the cluster. • The repressor protein is a tetramer of identical subunits coded by the gene lacI. Figure 12.5

7. Ex Biochem c12-operon 12.5 The lac Operon Can Be Induced • Small molecules that induce an operon are identical with or related to the substrate for its enzymes. • β-galactosides are the substrates for the enzymes coded by lacZYA. • In the absence of β-galactosides, the lac operon is expressed only at a very low (basal) level. • Addition of specific β-galactosides induces 誘發 transcription of all three genes of the operon. • The lac mRNA is extremely unstable; • as a result, induction can be rapidly reversed. • The same types of systems that allow substrates to induce operons coding for metabolic enzymes can be used to allow end-products to repress the operons that code for biosynthetic enzymes.

8. Ex Biochem c12-operon Figure 12.06: lac expression responds to inducer.

9. Ex Biochem c12-operon 12.6 Repressor Is Controlled by a Small Molecule Inducer • An inducer functions by converting the repressor protein into a form with lower operator affinity. • Repressor has two binding sites: • one for the operator • one another for the inducer • Repressor is inactivated by an allosteric interaction: • Binding of inducer at its site changes the properties of the DNA-binding site

10. Ex Biochem c12-operon Inducer of lac Operon • IPTG: common inducer for lac Operon used in lab • Similar structure to lactose • Although can NOT be digested by beta-galactosidase

11. Ex Biochem c12-operon Figure 12.07: A repressor tetramer binds the operator to prevent transcription. Figure 12.08: Inducer inactivates repressor allowing gene expression. http://www.youtube.com/watch?v=oBwtxdI1zvk

12. Ex Biochem c12-operon 12.7 cis-Acting Constitutive Mutations Identify the Operator • Mutations in the operator cause constitutive expression of all three lac structural genes. • These mutations are cis-acting and affect only those genes on the contiguous 連續的 stretch of DNA. • Cis-acting • Referring to a regulatory sequence in DNA (e.g., enhancer, promoter) that can control a gene only on the same chromosome. • In bacteria, cis-acting elements adjacent or proximal to the genes they control, whereas in eukaryotes they may also be far away Figure 12.9

13. Ex Biochem c12-operon 12.8 trans-Acting Mutations Identify the Regulator Gene • Mutations in the lacI gene: • are trans-acting • affect expression of all lacZYA clusters in the bacterium • trans-acting • Referring to DNA sequences encoding diffusible proteins (e.g., transcription activators and repressors) that control genes on different chromosomes • Mutations that eliminate lacI function: • cause constitutive expression • are recessive

14. Ex Biochem c12-operon 12.8 trans-Acting Mutations Identify the Regulator Gene • Mutations in the DNA-binding site of the repressor are constitutive because the repressor cannot bind the operator. • Mutations in the inducer-binding site of the repressor: • prevent it from being inactivated • cause uninducibility • Mutations in the promoter are: • uninducible • cis-acting Figure 12.10

15. Ex Biochem c12-operon 12.14 Repressor Protein Binds to the Operator • Repressor protein binds to the double stranded DNA sequence of the operator. • The operator is a palindromic sequence of 26 bp. Figure 12.17

16. Ex Biochem c12-operon Operons in eukaryotes • gene order in eukaryotes is NOT random. • numerous reports of gene clusters of related function in eukaryotes, even humans • significant tendency for genes from the same metabolic pathway to cluster. • Extensive clustering of non-homologous genes that are co-ordinately expressed in eukaryotes, including humans

17. Ex Biochem c12-operon Operons in eukaryotes • At the functional level, physical clustering may be advantageous because it allows groups of genes to be co-ordinately regulated at the levels of nuclear organization and/or chromatin. • The alleles could interact well by being co-localized in regions of chromosomes that facilitate co-ordinate regulation

18. Ex Biochem c12-operon Hurst, 2004

19. Ex Biochem c12-operon Osbourn, 2009