1. Regulation of Gene Expression AP Biology Ch 15

2. Gene  Protein Control Feedback inhibition – enough product is made the system shuts down – More product is made when needed – The product shuts down the process Gene Expression – genes are only expressed when needed. Often regulated at transcription.

3. Gene Expression: Prokaryotes Operon – grouped genes that are transcribed together – code for functionally similar proteins Key Players – Promoter – section of DNA where RNA polymerase binds – Operator – Controls activation of transcription on off switch between promoter and genes for proteins – structural genes – Repressor protein – binds to operator to block RNA polymerase and shut down transcription Turns off the operon Corepressor – keeps the repressor protein on the operator – Trp operon Inducer – pulls repressor off the operator – Turns on the operon – lactose on the lac operon – Regulatory gene – produces the repressor protein – Structural genes – code for proteins

4. Positive and Negative Gene Regulation Negative – Repressible: usually on but can be inhibited trp operon, allosteric inhibition, tryptophan present prevents its own production. (anabolic) – Inducible: usually off, but can be turned on, an inducer (a specific small molecule, allolactose in the lac operon) inactivates the repressor and allows transcription (catabolic) Positive – E. coli prefer to use glucose for energy, they will only use lactose when glucose is in short supply – glucose cAMP binds to regulatory protein “CAP” & stimulates gene transcription Positive gene regulation! – The cAMP & CAP combination allow RNA polymerase to bind to the promoter sequence more efficiently. – Remember cAMP is regulating the gene expression in the bacteria

5. Trp operon: repressible, always making tryptophan, repressed if tryptophan is “eaten” tryptophan is necessary for the cell to function

6. Lac operon: inducible, only turned on if lactose is “eaten” lactose is not necessary for the cell to function

7. Eukaryotic Chromosome Chromosomes – tightly coiled DNA around proteins during cell division Chromatin – loosely packed DNA around proteins Histones – protein which the DNA wraps around Nucleosomes – grouped histones together – Heterochromatin – tighter packed chromatin Not transcribing – Euchromatin – looser packed chromatin Transcription occurring

8. Gene Expression: Eukaryotes Cell Differentiation – cell specialization All cells contain the same genes The genes that are expressed determines the type of cell – Ex: Skin cell vs. a nerve cell

9. Chromatin Regulation Histone acetylation – allows transcription factors to bind to DNA allowing transcription to occur – Creates loosely packed DNA - euchromatin DNA Methylation – occurs after DNA synthesis has occurred – Lower transcription rates – One X in females is highly methylated – Works w/ a deacetylation enzyme in some spp.

10. Epigenetic inheritance Not controlled by base sequences. DNA methylation (deactivates one homologous chromosome) may explain abnormal or unexpected DNA expression as is often seen in identical twins. http://images.the-scientist.com/content/images/general/55342-1.jpg

11. Regulation of Transcription Transcription involves RNA Polymerase II and transcription factors RNA polymerase II attaches to the promoter (TATA box) sequence to begin transcription Control elements – non coding sequences of DNA where the transcription factors attach

12. Regulation of Transcription..\..\..\AP Bio 15-16\Genetics\15_10TranscripInitiation_A.swf..\..\..\AP Bio 15-16\Genetics\15_10TranscripInitiation_A.swf Enhancer – control element far from a gene or intron Activator – bind to enhancers to turn on transcription of a gene Transcription factors + enhancer + activator + RNA Polymerase II = transcription initiation complex – Needed for transcription to begin Repressors – inhibit gene expression – Turn off transcription – Block activators from binding to enhancers

13. Distal control element Activators Enhancer Promoter Gene TATA box General transcription factors DNA-bending protein Group of Mediator proteins RNA Polymerase II RNA Polymerase II RNA synthesis Transcription Initiation complex Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation A DNA-bending protein brings the bound activators closer to the promoter. Other transcription factors, mediator proteins, and RNA polymerase are nearby. 2 Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites. 1 The activators bind to certain general transcription factors and mediator proteins, helping them form an active transcription initiation complex on the promoter. 3

14. RNA Processing Regulation Alternative RNA Splicing – different regions of the pre-mRNA serve as introns or exons creating different mRNA strands depending on what is spliced out.

15. mRNA Degredation Prokaryotes – Short Life span – Degraded in seconds – Allows rapid response to environmental changes Eukaryotes – Survive from hours to weeks – Internal conditions constant, no need for rapid response

16. ncRNA: 1000’s of RNA’s, current research miRNA’s - micro RNA hat can degrade mRNA or block translation Causes mRNA to fold on itself and base pair to create dsRNA which is then digested with an enzyme Short interferring RNA (siRNA) – also degrade mRNA or block translation (blocking by siRNA is called RNAi, or RNA interferance)

17. Protein Degradation 18_12ProteinDegradation_A.swf 18_12ProteinDegradation_A.swf Proteosomes – break apart proteins in to smaller peptide units Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Ubiquitin Protein to be degraded Ubiquinated protein Proteasome and ubiquitin to be recycled Protein fragments (peptides) Protein entering a proteasome

18. Single Gene Expression Different cells express different genes, therefore they make different mRNA’s We can detect mRNA in a cell using nucleic acid hybridization, by pairing it to a nucleic acid probe Each probe is labeled with a fluorescent tag to allow visualization The technique allows us to see the mRNA in place (in situ) in the intact organism and is thus called in situ hybridization

19. Figure 15.16 mRNAs cDNAs Embryonic stages 1 cDNA synthesis PCR amplification Gel electrophoresis Results Technique 1 2 3 Primers  -globin gene 23456

20. Figure 15.15-5 Test tube containing reverse transcriptase and mRNA DNA in nucleus mRNAs in cytoplasm Reverse transcriptase makes the first DNA strand. Reverse transcriptase mRNA Poly-A tail DNA strand Primer 5 3 3 5 A A A 1 2 mRMA is degraded. 3 5 3 3 5 A A A DNA polymerase synthesizes the second strand. DNA polymerase 5 3 3 5 4 5 3 3 5 cDNA cDNA carries complete coding sequence without introns. 5 T TTTTT TTTT

21. Groups of Gene Expression Recall Microarray assays: Used to pinpoint differences in gene expression between 2 different cell types How it’s done: – Sequence a genome – Use PCR to copy the genes (verification steps here) – Split the genes into single strands – Place the single stranded DNA onto microscope slides in spots (robots & computers do all this)