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Lecture 14, Tues, Feb 24

Regulation of gene expression
•! Key points from Lecture 13 (mutations)
•! Briefly: promoters & transcription
•! Prokaryote regulation
•! inducible gene regulation: lac operon
–! lac repressor
–! CAP activator

•! repressible gene regulation: trp operon

•! Eukaryotic gene expression
•! promoters and enhancers

•! RNA interference
•! newly recognized method to regulate gene
expression
•! also useful genetic tool (medical tool?)

Klug
Chapter 15

Class business
•  Midterm grades uploaded to Ted
–  Reflects +7 added to grade that is written on paper
exam
–  Q6 regrade: for branched diagram, it is required to
show how you multiplied together the individual
phenotypes (or we also accepted genotypes)

•  Midterms available from Bonner 4th floor
–  Outside Bonner 4334 if waiver
–  Inside Bonner 4334 if no waiver

•  Professor Troemel office hours today
10-11am in Bonner 4202

Key points from Lecture 13
Mutant lecture: Ch. 14 in Klug
•! Mutations: chain of effects from DNA protein - function - dominant/recessive
•! Causes of mutations
–! internal/spontaneous
–! external/induced

•! DNA repair pathways
Chain of events from mutation to outcome
Genetic code will be provided on a test

Clicker Q1: What kind of mutation is this? •  TAT AAA CAT GAC to •  TAA AAA CAT GAC A) substitution. missense B) substitution. nonsense D) Insertion E) Frameshift . silent C) substitution.

What kind of mutation is this? •! TAT AAA CAT GAC to •! TAT GAA ACA TGA C –! missense –! nonsense –! silent –! deletion –! insertion –! frameshift What other problem does this frameshift cause? premature stop Asp to stop .

What kind of effect can this mutation have on protein function? •! Asp to stop –! gain of function? –! loss of function? •! What kind of loss of function might this be? –! what if stop happens at beginning of the protein? •! null (complete lof) Will this mutation likely be dominant or recessive? .

Tues. Feb 24 Regulation of gene expression •! Key points from Lecture 13 (mutations) •! Prokaryote regulation •! inducible gene regulation: lac operon –! lac repressor –! CAP activator •! repressible gene regulation: trp operon •! Eukaryotic gene expression •! promoters and enhancers •! RNA interference •! newly recognized method to regulate gene expression •! also useful genetic tool (medical tool?) Klug Chapter 15 .Lecture 14.

interactions mapping. regulation of amount •! some E. mutations and consequences •! But how are those genes regulated? –! Only certain genes are expressed at certain times in certain places in certain amounts –! e. while others are 100.g.000 copies per cell few protein copies many protein copies .How is gene expression regulated? •! Until now: gene transmission. coli proteins present at 5-10 molecules per cell.

g. cell-type specific expression: Remember in multicellular organisms. DNA for all genes is in all cells. but only EXPRESSED in certain cells pancreatic cell makes insulin retinal cell doesnt (even though it has the gene) pancreatic cell doesn’t make pigment retinal cell makes retinal pigment .Cell-type specific gene expression •! Only certain genes are expressed at certain times in certain places in certain amounts •! e.

How do organisms respond to changing environmental conditions? •! One mechanism of response is to regulate expression of mRNA from genomic DNA regulate this step probably predominant level of regulation in prokaryotes Figure 1-8 .

Transcription/promoters Repressor proteins Figure 12-8 Negative regulation of transcription .

What makes us different from chimps? •  Humans and chimps share ~98% of genes –  the major difference between us and chimps is likely to be gene regulation .

bacteria express enzymes to utilize those sugars •! only make enzymes when substrates are present •! Inducible enzymes (induced by substrate) •! Repressible system (end product) –! If tryptophan is present in the environment.Regulation of gene expression ON OFF flipping a switch •! Bacteria (and other microorganisms) adapt to changing environmental conditions •! Inducible system –! If certain sugars are present in the environment (e. lactose).g. it will repress enzymes that synthesize tryptophan •! only make enzymes when end-product is absent •! repressible enzymes (repressed by end-product) Can also have a combination of these systems .

Negative vs. positive control of transcription Negative regulation of transcription Lactose inhibits the repressor: Inducible system Repressor protein Lactose utilization genes .

Example of inducible gene regulation in bacteria: lac operon •  Genes are found in “operons” –  lac operon allows bacteria to utilize lactose •  several genes contained in this operon Figure 15-1 .

which cleaves lactose into galactose and glucose .Lactose is processed into galactose and glucose by lacZ gene Figure 15-2 lacZ encodes betagalactosidase enzyme.

lacZ (and lacY and lacA) only made when lactose is available .

regulated together •! Mutants isolated that cannot use lactose as carbon source X X Figure 15-1 lacZ. lacY.mutant lacY.mutant OFF . lacA transcribed together –! therefore.Constitutively OFF mutants •! Normally lac operon induced in the presence of lactose –! lacZ.

acts in trans .ON Constitutive ON lac mutants: lacI•! Key insights from regulatory mutants: –! constitutive mutant: operon is always ON •! lac enzymes expressed in absence of lactose X Figure 15-1 lacI is a repressor Constitutive mutant: lacI.

Constitutive ON lac mutants: lacOC •! Key insights from regulatory mutants: –! constitutive mutant: operon is always ON •! lac enzymes expressed in absence of lactose X Figure 15-1 Constitutive mutant: lacOC lacO is the operator acts in cis ON .

a promoter. transcription factor activator.g. operator •! trans-acting element –! factors (usually proteins) that control gene expression through a cis element •! e.g. repressor What controls these cis and trans acting elements? Environmental factors: lactose .cis vs. trans effects •! cis-acting element –! DNA sequence that regulates expression of a gene located nearby (on the same chromosome) •! e.

Lactose regulates the lac operon via the lacI repressor •  Lactose inhibits the repressor –  leads to induction of lac operon Figure 15-5 .

Lactose regulates the lac operon via the lacI repressor Figure 15-5 Normal regulation no lactose: OFF .

Lactose regulates the lac operon via the lacI repressor Figure 15-5 ON Normal regulation Lactose flips the switch to ON .

Allosteric regulation •  Lactose causes a conformational change in lacI repressor so that it can no longer bind to the operator Gray blob (lac I repressor) undergoes conformational change .

lac operon constitutively ON when repressor or operator is defective Figure 15-6 Defective repressor ON .

lac operon constitutively ON in absence of repressor or operator Figure 15-6 Defective operator ON .

and I+? .can test this with merozygote Diploid for certain genes: can ask about dominant/recessive What would happen if you combined I.Phenotypes of lac mutants Testing the model! no enzyme Zno repressor Ioperator constitutively on OC Model makes predictions .

Clicker Q2: What would happen if you combined I.and I+? ? ? A: B: + + + - C: - - D: - + .

and I+? B) Rescues .Phenotypes of lac mutants cis/trans elements no enzyme Zno repressor Ioperator constitutively on OC What would happen if you combined I.looks like wild-type. I+ is dominant What would happen if you combined OC and O+? .

Clicker Q3: What would happen if you combined O+ and OC? A: ? + ? B: + + - C: - - D: - + .

and I+? B) Rescues . OC is dominant .Phenotypes of lac mutants cis/trans elements no enzyme Zno repressor Ioperator constitutively on OC What would happen if you combined I.looks like wild-type What would happen if you combined OC and O+? A) Constitutively ON. like OC alone.

Phenotypes of lac mutants no enzyme Zno repressor Ioperator constitutively on OC Review other predictions in your textbook .

youtube.com/watch? v=W6s7I3I0Kh4&feature=related .Video of lac operon •  http://www.youtube.com/watch? v=iPQZXMKZEfw •  http://www.

trans effects •  cis-acting element –  DNA sequence that regulates expression of a gene located nearby (on the same chromosome) •  e. a promoter.g. repressor .g.cis vs. transcription factor activator. operator •  trans-acting element –  factors (usually proteins) that control gene expression through a cis element •  e.

D) production of nonfunctional βgalactosidase. E) no change in the expression of lac. as the repressor cannot bind.Clicker Q4: A mutation in the P region of the lac operon would most likely result in A) constitutive expression of lac. C) reduced expression of lac. B) constitutive expression of lac. . as lactose cannot bind.

.Clicker Q4: A mutation in the P region of the lac operon would most likely result in Answer: C. Explanation: The promoter (P) allows RNA polymerase to bind and begin transcription of lac. reduced expression of lac. A change in P would be likely to affect RNA polymerase binding and reduce lac transcription.

What do E. coli do if they have BOTH lactose and glucose? Lactose is converted to galactose and glucose Galactose is converted to glucose Glucose is a preferred carbon source How is this controlled? Figure 15-2 .

which makes cAMP .Another regulator of the lac operon: CAP •  Catabolite activating protein (CAP) –  activates expression of lac operon •  binds to promoter –  CAP binds promoter better with cAMP •  cAMP levels are lower in presence of glucose –  glucose inhibits adenylate cyclase.

Another regulator of the lac operon: CAP Figure 15-8 .

Another regulator of the lac operon: CAP Figure 15-8 Combination of positive and negative regulation determines lac operon transcription .

Clicker Q5: The lac operon is normally expressed: A) in the absence of both lactose and glucose. B) in the absence of lactose and the presence of glucose. . D) in the presence of both lactose and glucose. E) None of the above. C) in the presence of lactose and the absence of glucose.

. The absence of glucose allows CAP to form a complex with cAMP. and facilitate RNA polymerase binding and lac transcription. in the presence of lactose and the absence of glucose. the presence of lactose is necessary to inactivate the repressor. Explanation: For lac expression. bind to the CAP site.Clicker Q5: The lac operon is normally expressed: Answer: C.

More than ever. and that would persist as long as they exist. research seemed to be identified with human nature! It was by far the best means found by man to face the chaos of the universe.54th anniversary of the lac operon •! Jacob & Monod elucidated this regulation –! Celebrated 3 years ago in the journal Science where Jacob reflected on their findings: •! Thus did we discover a mechanism fundamental to all living beings from their very beginnings. Success of night science modern lab technique: blue/white selection ...

Gene regulation •  can have repressors and activators –  sometimes several of each •  Combinatorial control .

Tues.Lecture 14. Feb 24 Regulation of gene expression •! Key points from Lecture 13 (mutations) •! Prokaryote regulation •! inducible gene regulation: lac operon –! lac repressor –! CAP activator •! repressible gene regulation: trp operon •! Eukaryotic gene expression •! promoters and enhancers •! RNA interference •! newly recognized method to regulate gene expression •! also useful genetic tool (medical tool?) Klug Chapter 15 .

Example of repressible gene regulation in bacteria: trp operon •  Energetically favorable to repress gene expression for tryptophan (trp) biosynthesis if trp is available from media Figure 15-9 .

Example of repressible gene regulation in bacteria: trp operon •  Energetically favorable to repress gene expression for tryptophan (trp) biosynthesis if trp is available from media Figure 15-9 .

ON Example of repressible gene regulation in bacteria: trp operon •! Absence of tryptophan Figure 15-9 .

Example of repressible gene
regulation in bacteria: trp operon
•! Presence of tryptophan

Figure 15-9

OFF

Lecture 14, Tues, Feb 24
Regulation of gene expression
•! Key points from Lecture 13 (mutations)
•! Prokaryote regulation
•! inducible gene regulation: lac operon
–! lac repressor
–! CAP activator

•! repressible gene regulation: trp operon

•! Eukaryotic gene expression
•! promoters and enhancers

•! RNA interference
•! newly recognized method to regulate gene
expression
•! also useful genetic tool (medical tool?)

Klug
Chapter 15

Gene regulation is more
complicated in eukaryotes
•! Gene expression has to be regulated in
amount, time AND place

pancreatic cell makes insulin

retinal cell doesnt
(even though it has the gene)

Animal cell Figure 2-1 * more cell organization: e.g.g.Eukaryote vs. ER. mitochondria. nucleus. Prokaryote e. Golgi * Usually diploid and several linear chromosomes * DNA packaged with histones Bacterial cell Figure 2-2 * Haploid and one circular chromosome BOTH have doublestranded DNA! .

it will be! Figure 15-11 .Gene regulation can occur at many stages in eukaryotes Rule in biology: if it can be regulated.

Transcription in eukaryotes can be regulated by cis and trans elements .

Eukaryotic gene regulation at level of cis-acting elements •! Promoters are adjacent to genes –! specify basal expression of genes •! Enhancers are farther away Can alter transcription of genes .

Regulation at level of trans-acting elements •! Trans-acting elements bind to the cisacting elements Human metallothionein IIA gene promoter Figure 15-16 Combinatorial control .

locations. can make 2 different hormones with different structures.Regulation of mRNA splicing Due to alternative splicing. and functions! Figure 15-19 .

Can also have regulation of mRNA transport. translation into protein. etc. etc Figure 15-11 . stability.

Feb 24 Regulation of gene expression •! Key points from Lecture 13 (mutations) •! Prokaryote regulation •! inducible gene regulation: lac operon –! lac repressor –! CAP activator •! repressible gene regulation: trp operon •! Eukaryotic gene expression •! promoters and enhancers •! RNA interference •! newly recognized method to regulate gene expression •! also useful genetic tool (medical tool?) Klug Chapter 15 . Tues.Lecture 14.

RNA interference: a newly recognized mode of gene regulation •! Shaking up the central dogma dsRNA ~21-24 nucleotides .

How was RNAi discovered? (and why was it missed for so long?) •! Fire and Mello injected unc-22 RNA into C. both blocked function. dsRNA elegans •! Expected antisense RNA to block function and sense to not block function •! instead. 2006 . and dsRNA blocked it best of all! Nature 1998 paper Nobel prize in medicine.

Mechanisms of RNAi •  Still being worked out .models likely to change •  Important to know what RNAi IS and how it is useful –  perhaps exists for defense against viruses –  useful for genetic studies in lab –  also may be a therapeutic in the clinic •  several companies focusing on this… .

Gene regulation by RNAinduced gene silencing Figure 15-21 .

Lecture 14. Feb 24 Regulation of gene expression •! Key points from Lecture 13 (mutations) •! Prokaryote regulation •! inducible gene regulation: lac operon –! lac repressor –! CAP activator •! repressible gene regulation: trp operon •! Eukaryotic gene expression •! promoters and enhancers •! RNA interference •! newly recognized method to regulate gene expression •! also useful genetic tool (medical tool?) Klug Chapter 15 . Tues.