2. AP Biology Lecture #35 Gene Regulation

3. Positive gene control occurs when an activator molecule interacts directly with the genome to switch transcription on. Even if the lac operon is turned on by the presence of allolactose, the degree of transcription depends on the concentrations of other substrates. The cellular metabolism is biased toward the utilization of glucose.

4. Positive Gene Regulation Some operons are also subject to positive control through a stimulatory protein, such as catabolite activator protein (CAP), an activator of transcription When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription

5. Positive Gene Regulation – If glucose levels are low (along with overall energy levels), then cyclic AMP (cAMP) binds to cAMP receptor protein (CRP) which activates transcription. If glucose levels are sufficient and cAMP levels are low (lots of ATP), then the CRP protein has an inactive shape and cannot bind upstream of the lac promotor.

6. 2007-2008 Control of Eukaryotic Genes

7. The BIG Questions… How are genes turned on & off in eukaryotes? How do cells with the same genes differentiate to perform completely different, specialized functions?

8. Evolution of gene regulation Prokaryotes – single-celled – evolved to grow & divide rapidly – must respond quickly to changes in external environment exploit transient resources Gene regulation – turn genes on & off rapidly flexibility & reversibility – adjust levels of enzymes for synthesis & digestion

9. Evolution of gene regulation Eukaryotes – multicellular – evolved to maintain constant internal conditions while facing changing external conditions homeostasis – regulate body as a whole growth & development – long term processes specialization – turn on & off large number of genes must coordinate the body as a whole rather than serve the needs of individual cells

10. Points of control The control of gene expression can occur at any step in the pathway from gene to functional protein 1.packing/unpacking DNA 2.transcription 3.mRNA processing 4.mRNA transport 5.translation 6.protein processing 7.protein degradation

11. 1. DNA packing as gene control Degree of packing of DNA regulates transcription – tightly wrapped around histones no transcription genes turned off  heterochromatin darker DNA (H) = tightly packed  euchromatin lighter DNA (E) = loosely packed H E

12. DNA methylation Methylation of DNA blocks transcription factors – no transcription  genes turned off – attachment of methyl groups (–CH 3 ) to cytosine C = cytosine – nearly permanent inactivation of genes ex. inactivated mammalian X chromosome = Barr body

13. Histone acetylation Acetylation of histones unwinds DNA  loosely wrapped around histones enables transcription genes turned on  attachment of acetyl groups (–COCH 3 ) to histones conformational change in histone proteins transcription factors have easier access to genes

14. Epigenetic Inheritance Although the chromatin modifications just discussed do not alter DNA sequence, they may be passed to future generations of cells The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance

15. 2. Transcription initiation Control regions on DNA – promoter nearby control sequence on DNA binding of RNA polymerase & transcription factors “base” rate of transcription – enhancer distant control sequences on DNA binding of activator proteins “enhanced” rate (high level) of transcription

16. Model for Enhancer action Enhancer DNA sequences – distant control sequences Activator proteins – bind to enhancer sequence & stimulates transcription Silencer proteins – bind to enhancer sequence & block gene transcription Turning on Gene movie

17. Transcription complex Enhancer Activator Coactivator RNA polymerase II A B F E H TFIID Core promoter and initiation complex Activator Proteins regulatory proteins bind to DNA at distant enhancer sites increase the rate of transcription Coding region T A Enhancer Sites regulatory sites on DNA distant from gene Initiation Complex at Promoter Site binding site of RNA polymerase

18. Fig. 18-9-3 Enhancer TATA box Promoter Activators DNA Gene Distal control element Group of mediator proteins DNA-bending protein General transcription factors RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis

19. 3. Post-transcriptional control Alternative RNA splicing – variable processing of exons creates a family of proteins

20. 4. Regulation of mRNA degradation Life span of mRNA determines amount of protein synthesis – mRNA can last from hours to weeks RNA processing movie

21. 5. Control of translation Block initiation of translation stage – regulatory proteins attach to 5' end of mRNA prevent attachment of ribosomal subunits & initiator tRNA block translation of mRNA to protein Control of translation movie

22. 6-7. Protein processing & degradation Protein processing – folding, cleaving, adding sugar groups, targeting for transport Protein degradation – ubiquitin tagging – proteasome degradation Protein processing movie

23. Ubiquitin “Death tag” – mark unwanted proteins with a label – 76 amino acid polypeptide, ubiquitin – labeled proteins are broken down rapidly in "waste disposers" proteasomes 1980s | 2004 Aaron Ciechanover Israel Avram Hershko Israel Irwin Rose UC Riverside

24. Proteasome Protein-degrading “machine” – cell’s waste disposer – breaks down any proteins into 7-9 amino acid fragments cellular recycling play Nobel animation

25. Concept 18.3: Noncoding RNAs play multiple roles in controlling gene expression Only a small fraction of DNA codes for proteins, rRNA, and tRNA A significant amount of the genome may be transcribed into noncoding RNAs Noncoding RNAs regulate gene expression at two points: mRNA translation and chromatin configuration

26. RNA interference Small interfering RNAs (siRNA) – short segments of RNA (21-28 bases) bind to mRNA create sections of double-stranded mRNA “death” tag for mRNA – triggers degradation of mRNA – cause gene “silencing” post-transcriptional control turns off gene = no protein produced NEW! siRNA

27. Action of siRNA siRNA double-stranded miRNA + siRNA mRNA degraded functionally turns gene off Hot…Hot new topic in biology mRNA for translation breakdown enzyme (RISC) dicer enzyme

28. initiation of transcription 1 mRNA splicing 2 mRNA protection 3 initiation of translation 6 mRNA processing 5 1 & 2. transcription - DNA packing - transcription factors 3 & 4. post-transcription - mRNA processing - splicing - 5’ cap & poly-A tail - breakdown by siRNA 5. translation - block start of translation 6 & 7. post-translation - protein processing - protein degradation 7 protein processing & degradation 4 4 Gene Regulation

29. Molecular Biology of Cancer Oncogene cancer-causing genes Proto-oncogene normal cellular genes How? 1- movement of DNA; chromosome fragments that have rejoined incorrectly 2- amplification; increases the number of copies of proto-oncogenes 3-proto-oncogene point mutation; protein product more active or more resistant to degradation Tumor-suppressor genes changes in genes that prevent uncontrolled cell growth (cancer growth stimulated by the absence of suppression)

30. Cancers result from a series of genetic changes in a cell lineage – The incidence of cancer increases with age because multiple somatic mutations are required to produce a cancerous cell – As in many cancers, the development of colon cancer is gradual

31. 2007-2008 Turn your Question Genes on!