Lecture 18 June 2 nd, Gene Expression Regulation Mutations

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1 Lecture 18 June 2 nd, 2016 Gene Expression Regulation Mutations

2 From Gene to Protein Central Dogma Replication DNA RNA PROTEIN Transcription Translation RNA Viruses: genome is RNA Reverse Transcriptase (RT) DNA Protein Inserts into host (e.g. human cell) genome (INTEGRATION) mrna RT cdna (Complementary DNA)

3 From Gene to Protein: Mutations DNA* RNA* PROTEIN* Mutation are changes in genetic material of a cell. Chromosomal Mutations: (e.g. Duplications and deletions) large-scale mutations: multiple genes are affected Multiple phenotypes Point Mutations: 1 base pair (bp) substitution in one gene Missense mutation 1 bp change different a.a. encoded UGC (cys) UGG (trp) Nonsense mutation 1 bp change STOP codon UGC (cys) UGA (STOP) Silent mutation 1 bp change codon is the same (due to wobble) UGC (cys) UGU (cys)

4 From Gene to Protein: Mutations DNA* RNA* PROTEIN* Frameshift Mutations: Insert or Delete a basepair Frameshift in ORF Frameshift in ORF Insert or delete an a.a.

5 From Gene to Protein: Mutations (silent)

6 From Gene to Protein: Mutations What about mutations that are not in the coding sequence? Affect mrna synthesis - promoter - termination - splicing Affect Translation - mutation in RBS (Prok) - Stability of mrna (3 UTR polyaaa tail)

7 Regulation of Gene Expression Every cell in the body has the same DNA So how can cells be so different from one another?? (cell shape, function, etc) Transcription Gene Regulation!!! DNA RNA

8 Regulation of Gene Expression Transcriptional UNIT (DNA!) Promoter 1 st base of mrna Terminator P Start Stop T 5 UTR Coding Region 3 UTR Promoter is very important RNA polymerase binds choses sense/template strand Can Determine how active a gene will be!!

9 Regulation of Gene Expression Promoters DNA sequence that RNA polymerase binds Good Promoter Poor Promoter RNA Pol binds tightly and lots of mrna is made RNA Pol binds poorly and little of mrna is made Encoded in the DNA of the promoter how often a gene is transcribed Promoter sequences are constantly changing from: good to poor HOW? Allosteric Regulation!! Regulation of RNA polymerase Transcriptional Activator: Recruit RNA polymerase to a promotoer: ON! Transcriptional Repressor: Block RNA polymerase: OFF!

10 Regulation of Gene Expression Transcriptional Activators and Repressors Repressor (TR) can bind at the promoter or a nearby sequence and block RNA pol : turns gene OFF! RNA Pol P ORF

11 Regulation of Gene Expression Transcriptional Activators and Repressors Repressor (TR) can bind at the promoter or a nearby sequence and block RNA pol : turns gene OFF! RNA Pol TR P X ORF RNA Pol P TR X ORF TR RNA Pol P X Binding site for repressor ORF

12 Regulation of Gene Expression Transcriptional Activators and Repressors Activator (TA) Binds to specific sequence, next to promoter, recruits RNA pol to that promoter turns gene ON! TA RNA Pol P ORF Binding site for activator

13 Regulation of Gene Expression Prokaryotic Gene Regulation A typical scenario: An individual bacteria can respond to environmental change by regulating gene expression How can negative feedback inhibition affect more than one gene?

14 Regulation of Gene Expression: Prokaryotic Prokaryotic Gene Regulation Normal food is glucose; Lactose is polymer of galactose-glucose so the amount of lactose permease (a receptor molecule which brings lactose inside cell) is normally low at the cell surface Lactose ( ) is low Lactose ( ) is high Allosteric Regulation! Bacteria will induce multiple genes which take advantage of lactose as a food source

15 Regulation of Gene Expression: Prokaryotic Prokaryotic Gene Regulation: OPERONS The Lac OPERON: Inducible Operon lacz: lacy: laca: Encodes beta-galactosidase (cleaves galactose-glucose into separate molecules) Encodes lactose permease (transports lactose inside cell) Encodes lactose transacetylase (helps cell use lactose) Operator: Controls RNA Pol access to gene!!

16 Regulation of Gene Expression: Prokaryotic Prokaryotic Gene Regulation: OPERONS The Lac OPERON: Inducible Operon Gene for repressor is adjacent to Lac operon!

17 Regulation of Gene Expression: Prokaryotic Prokaryotic Gene Regulation: OPERONS The Lac OPERON: Inducible Operon When Lactose is absent: default is OFF! No Transcription!

18 Regulation of Gene Expression: Prokaryotic Prokaryotic Gene Regulation: OPERONS The Lac OPERON: Inducible Operon When Lactose is present: ON! Lactose Binds repressor Transcription happens!

19 Regulation of Gene Expression: Prokaryotic Prokaryotic Gene Regulation: OPERONS The Tryptophan OPERON: Repressible Operon When Tryptophan is absent: default is ON! Transcription happens!

20 Regulation of Gene Expression: Prokaryotic Prokaryotic Gene Regulation: OPERONS The Tryptophan OPERON: Repressible Operon When Tryptophan is present: OFF! No Transcription!

21 Regulation of Gene Expression: Eukaryotes Organization of the Eukaryotic Genome Compared to simple Prokaryotes Much Larger Genome Multicellular Cell specialization (due to cell differentiation?) Has > 25,000 Genes Lots of DNA that does not code for RNA or protein KEY! Multiple levels of Gene Expression Regulation!

22 Regulation of Gene Expression: Eukaryotes Eukaryotic Gene Regulation No Operons 3 polymerases RNA pol I, II, and III RNA Pol s can t bind DNA alone Require Transcription factors!! DNA Promoter sequences don t act alone Proximal Control Sequence Enhancer Sequences Silencer Sequences Chromatin regulation: access to gene by RNA polymerase

23 Regulation of Gene Expression: Eukaryotes Transcription factors Bind to the minor and major grooves of the DNA Double helix Transcription factor DNA binding motifs (examples) DNA Sequence Specific!! Recruit RNA pol turn genes ON! Zinc helps stabilize a finger like protrusion that binds DNA at the major groove Helix-turn-Helix Zinc finger

24 Regulation of Gene Expression: Eukaryotes DNA Regulatory Elements (sequences!) Proximal Control Elements right next to promoter sequence transcription factors bind here allow for multiple genes in one pathway to be turned on HOW? Can share similar DNA Seq. in front of promoters for different genes Example: SRE sequences (stress response element) transcription factors will bind SRE element and turn multiple genes

25 Regulation of Gene Expression: Eukaryotes DNA Regulatory Elements (sequences!) Enhancers Transcription activators DNA sequences sometimes 1000 s of by away Recruit transcription factors turn genes ON! Bend DNA to bring activators near transcription factors and RNA pol

26 Regulation of Gene Expression: Eukaryotes Enhancers (transcription activators)

27 Regulation of Gene Expression: Eukaryotes DNA Regulatory Elements (sequences!) Enhancers Transcription activators DNA sequences sometimes 1000 s of by away Recruit transcription factors turn genes ON! Bend DNA to bring activators near transcription factors and RNA pol Silencers Transcription repressors 1000 s bp away inhibit transcription turn genes OFF!

28 Regulation of Gene Expression: Eukaryotes Chromatin DNA-Protein (Histones) complex DNA packing Nucleosome basic unit of DNA packing DNA wrapped around histones supercoiling DNA double Helix 2 M Histones 10 M Beads on a string (TEM) Linker DNA (string) Nucleosome Note: Chromosome (in metaphase of cell cycle) can be 1-2 microns in diameter!!

29 Regulation of Gene Expression: Eukaryotes How Can DNA Packing affect Gene Expression? Physically hide genes Affects accessibility! Block access to RNA Polymerase Transcription factors must break DNA-Histone interactions and recruit RNA Pol.

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