genetics lecture 14 2015-02-24

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, silent C) substitution, nonsense D) Insertion E) Frameshift

What kind of mutation is this?

What other problem does this frameshift cause?

! TAT AAA CAT GAC to ! TAT GAA ACA TGA C

! missense ! nonsense ! silent ! deletion ! insertion ! frameshift

Asp to stop What other problem does this frameshift cause? premature 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?


! Key points from Lecture 13 (mutations) ! Prokaryote regulation






Klug Chapter 15

How is gene expression regulated? ! Until now: gene transmission, interactions

mapping, mutations and consequences

! But how are those genes regulated? ! Only certain genes are expressed at certain times

in certain places in certain amounts ! e.g. regulation of amount

! some E. coli proteins present at 5-10 molecules per cell, while others are 100,000 copies per cell

few protein copies many protein copies

Cell-type specific gene expression ! Only certain genes are expressed at certain times in

certain places in certain amounts ! e.g. cell-type specific expression: Remember in multi-

cellular organisms, DNA for all genes is in all cells, but only EXPRESSED in certain cells

retinal cell makes retinal pigment pancreatic cell doesnt make pigment

pancreatic cell makes insulin retinal cell doesnt

(even though it has the gene)

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

Figure 12-8

Repressor proteins

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

Regulation of gene expression

! Bacteria (and other microorganisms) adapt to changing environmental conditions

! Inducible system ! If certain sugars are present in the environment (e.g.

lactose), 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, 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

OFF

ON

flipping a switch

Negative vs. positive control of transcription

Lactose utilization genes

Repressor protein

Lactose inhibits the repressor:

Inducible system

Negative regulation of transcription

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

Lactose is processed into galactose and glucose by lacZ

gene

lacZ encodes beta-galactosidase enzyme,

which cleaves lactose into galactose and glucose

Figure 15-2

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

Constitutively OFF mutants

lacZ- mutant

X

lacY- mutant

X

OFF

! Normally lac operon induced in the presence of lactose ! lacZ, lacY, lacA transcribed together ! therefore, regulated together

! Mutants isolated that cannot use lactose as carbon source

Figure 15-1

Constitutive ON lac mutants: lacI-

! Key insights from regulatory mutants: ! constitutive mutant: operon is always ON

! lac enzymes expressed in absence of lactose

lacI is a repressor acts in trans Constitutive mutant: lacI-

X

ON

Figure 15-1

Constitutive ON lac mutants: lacOC

! Key insights from regulatory mutants: ! constitutive mutant: operon is always ON

! lac enzymes expressed in absence of lactose

lacO is the operator acts in cis Constitutive mutant: lacOC

X

ON

Figure 15-1

cis vs. trans effects

! cis-acting element ! DNA sequence that regulates expression of a

gene located nearby (on the same chromosome) ! e.g. a promoter, operator

! trans-acting element ! factors (usually proteins) that control gene

expression through a cis element ! e.g. transcription factor activator, repressor

What controls these cis and trans acting elements? Environmental factors: lactose

Lactose regulates the lac operon via the lacI repressor

Lactose inhibits the repressor leads to induction of lac operon

Figure 15-5


Normal regulation no lactose:

OFF

Figure 15-5


Normal regulation

ON

Lactose flips the switch to ON

Figure 15-5

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

Defective repressor

ON

Figure 15-6

Defective operator

lac operon constitutively ON in absence of repressor or operator

ON

Figure 15-6

Phenotypes of lac mutants

no enzyme Z- no enzyme Zno repressor I- no repressor

operator constitutively on

OC

Testing the model!

Model makes predictions - can test this with merozygote

Diploid for certain genes: can ask about dominant/recessive

What would happen if you combined I- and I+?

Clicker Q2: What would happen if you combined I- and I+?

+ + + -

-

-

-

+

A: B:

C:

D:

? ?


no enzyme no enzyme no repressor no repressor


no repressor Z-

I- OC

cis/trans elements


B) Rescues - 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:

? ?



Z- I-

OC

cis/trans elements


B) Rescues - looks like wild-type

What would happen if you combined OC and O+? A) Constitutively ON, like OC alone, OC is dominant

no repressor

I




Z- I-

OC

Review other predictions in your textbook

no repressor operator constitutively on

Video of lac operon

http://www.youtube.com/watch?v=iPQZXMKZEfw

http://www.youtube.com/watch?v=W6s7I3I0Kh4&feature=related

cis-acting element DNA sequence that regulates expression of a

gene located nearby (on the same chromosome) e.g. a promoter, operator

trans-acting element factors (usually proteins) that control gene

expression through a cis element e.g. transcription factor activator, repressor

cis vs. trans effects

Clicker Q4: A mutation in the P region of the lac operon would

most likely result in A) constitutive expression of lac, as the

repressor cannot bind. B) constitutive expression of lac, as

lactose cannot bind. C) reduced expression of lac. D) production of nonfunctional -

galactosidase. E) no change in the expression of lac.

Answer: C. reduced expression of lac.

Explanation: The promoter (P) allows RNA polymerase to bind and begin transcription of lac. A change in P would be likely to affect RNA polymerase binding and reduce lac transcription.

Clicker Q4: A mutation in the P region of the lac operon would most

likely result in

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

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, which makes

cAMP


Figure 15-8


Combination of positive and negative regulation determines lac operon transcription

Figure 15-8

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. C) in the presence of lactose and the absence of glucose. D) in the presence of both lactose and glucose. E) None of the above.

Answer: C. in the presence of lactose and the absence of glucose.

Explanation: For lac expression, the presence of lactose is necessary to inactivate the repressor. The absence of glucose allows CAP to form a complex with cAMP, bind to the CAP site, and facilitate RNA polymerase binding and lac transcription.

Clicker Q5: The lac operon is normally expressed:

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, and that would persist as long as they exist... More than ever, 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.

Success of night science

modern lab technique: blue/white selection

Gene regulation

can have repressors and activators sometimes several of each

Combinatorial control








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 ! Absence of tryptophan

ON

Figure 15-9

Example of repressible gene regulation in bacteria: trp operon

! Presence of tryptophan

OFF

Figure 15-9








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)

Eukaryote vs. Prokaryote e.g. Animal cell Bacterial cell

* more cell organization: e.g. nucleus, mitochondria, ER, Golgi

* Usually diploid and several linear chromosomes

* Haploid and one circular chromosome

* DNA packaged with histones

BOTH have double-stranded DNA!

Figure 2-1 Figure 2-2

Gene regulation can occur at many stages

in eukaryotes

Rule in biology: if it can be regulated, it will be!

Figure 15-11

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 cis-acting elements

Human metallothionein IIA gene promoter

Combinatorial control Figure 15-16

Regulation of mRNA splicing

Due to alternative splicing, can make 2 different hormones with different structures, locations, and functions!

Figure 15-19

Can also have regulation of mRNA transport, stability, translation into protein, etc, etc

Figure 15-11








Klug Chapter 15

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. elegans

! Expected antisense RNA to block function and sense to not block function

! instead, both blocked function, and dsRNA blocked it best of all! Nature 1998 paper

dsRNA

Nobel prize in medicine, 2006

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 RNA-induced gene silencing

Figure 15-21








Klug Chapter 15

genetics lecture 14 2015-02-24

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