EXAM2_L18_L19_Control_Gene_Expression Flashcards Preview

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Flashcards in EXAM2_L18_L19_Control_Gene_Expression Deck (28)
1

PROKARYOTES:
if no glucose available, what is used for energy?
What happens if it is the only carbon source?

Lactose (glucose + galactose)

Three genes from LAC OPERON:
1. B-galactosidase (cleaves lactose)

2. Lactose Permease (transports lactose cyto membrane)

3. B-Galactoside Transacetylase (tx acetyl from AcetylCoa to B-galactoside)

2

Lac Operon Structure

Promoter(PI)/Repressor(I)/CAP/pLac/Operator||| Z/Y/A genes
Z- b-galactosidase
Y- Permease
A- Transacetylase

3

Positive control of lac operon:

When does it happen?
What are the 5 steps?

CAP: "catabolite activator protein"

No glucose-> lactose metabolism via CAP-cAMP

1. adenylyl cyclase activated (low glucose)
2. [cAMP] increases
3. cAMP binds CAP
4. CAP-cAMP binds DNA @ CAP SITE
5. RNApol binds promoter & makes 3 genes of lactose metabolism

4

When is cAMP high? Low?
What is CAP?
Why is cAMP so important?
What happens if there is high cAMP?
Sequence of 4 events?

LOW glucose= HIGH cAMP
HIGH glucose = LOW cAMP

CAP: "catabolite activator protein"

CAP can only bind to DNA if cAMP present

1. if there is high cAMP that means no glucose & lac operon activation needed.
2. cAMP binds CAP, CAP binds DNA,
3. RNApol recruited & makes 3 lac genes,
4. lac operon/metabolism ACTIVE

5

Negative Control of "lac I gene" & "lac repressor"

When is the lac operator inhibited?
What inhibits?
Why?
What makes repressor?

Lac OPERATOR inhibited-- if NO LACTOSE
-REPRESSOR binds/inhibits OPERATOR (if no lactose)

*The only reason to TURN ON lac operator is if there is lactose to be metabolized

"lac I" gene encodes "lac repressor"
lac I- always present in cells (constitutively expressed)

6

lac I gene and lac repressor activity:

What happens if cell has LACTOSE?
What needed for RNApol to bind and transcribe?

Lactose>> allolactose "inducer"

allolactose binds/inhibits repressor.
-->OPERATOR now active

If cAMP--CAP binds DNA--DNApol binds-->Transcription

7

What two ways is the lac operon negatively controlled?

1. Lac repressor binding operator when no lactose
-repressor constitutively available
-binds regardless of glucose or CAP

2. No CAP-cAMP binding to CAPsite
-CAP only binds if cAMP available (no glucose)

8

When will lac operon be active?

ONLY ACTIVE IF:
1. no glucose (cAMP binds CAP-> binds DNA)
2. has lactose (repressor disabled/releases from operator)

RNApol can bind and transcribe at START site

9

Differences in Eukaryote Transcriptional regulation:

What are two levels of control of gene expression?

1. no Operons
2. complex (has nucleus thus separate areas of transcription/translation)

1. Short Term Control (daily needs on/off quickly)
2. Long Term Control (gene reg in development/differentiation)

10

What are 6 ways to regulate Eukaryotic gene expression?

1*. transcription (activate/repress)
2. RNA processing (alternative splicing)
3. mRNA transport (stuck in nucleus=degraded)
4*. mRNA Translation (rate & re-initiation: 5'cap/polyA tail)
5. mRNA degradation (Long polyA tail= longer life)
6. Protein activity/degredation (post translat modification)

11

CIS (cis-acting elements)
TRANS Elements (trans-acting elements)

CIS-- ENHANCER SEQUENCE ON DNA (regulatory)

TRANS- An ACTIVATOR PROTEIN BINDS CIS SEQUENCE


CIS Elements= Regulatory DNA seq. where TF's bind
--- ie:(promoters/enhancers)

TRANS- gene regulatory proteins that recognize cis elements.
----ie: (TFIID, Activators)

12

Eukaryotic Gene Transcription Regulation:
GTF's

General transcription factors- REQUIRED FOR INITIATION
- bind to promoter region of a gene

13

What are activators, enhancers, transcription factors?
What do they do?
How do they work?

ACTIVATORS (TF's)- Bind to a DNA enhancer sequence to increase transcription. VIA signals

Activators bind enhancer and then they can regulate RNApol II- - Increases efficiency/rate regardless of distance or position

14

If Enhancer is over 1000bp's away how can they activate transcription at the START SITE?

DNA Looping (brings the two regions close together)

15

Two domains of Trans-acting factors
What are the roles of each?

1. DNA binding domain- (binds dna) at motif

2. Activation domain:
-binds GTF's and co-activators
-Modifies chromatin structure

16

What are 4 structural motifs commonly found in the DNA-binding domains of regulatory transcription factors?

1. Zinc-finger
2. Leucine Zipper
3. Helix-loop-Helix
4. Helix-turn-helix

17

Zinc Finger

binding domain example?
Cis-Element?

Protein bind Zn ions & form loops/fingers

IE: Steroid hormone receptor (estrogen)

-Cis-element: HRE (hormone response element)

18

Leucine Zipper Motif

binding domain example?
Cis-Element?

2 alpha helix w/ leucines facing each other along length of helix

-Dimerization allows protein to bind on both sides of double helix

Ex: CREB (cAMP response element binding protein)
cis-element: CRE

19

Helix-loop-Helix motif

binding domain example?
Cis-Element?

DNA-binding domains formed by dimerization of two polypeptide chains

Ex: transcription factor MYC
cis-element: E-BOX

20

Helix-Turn-Helix Motif
binding domain example?
Cis-Element?

2 helix separated by turn (a turn is longer than a loop)
- one helix binds, 2nd helix stabilizes by lying across it

Ex: Homeodomain (HOX) proteins
- embryonic development (CNS, axial skeleton, etc)

cis-element: HOMEOBOX

21

PIT-1 TF
What does it encode?
What genes does it transcribe?
What happens if misregulated?
What happens if pit1 is low?
How do you treat children?

Pituitary gland makes-->Growth Hormone

PIT-1 gene encodes PIT-1 TF
PIT-1 TF required for transcribing GH & PROLACTIN genes (and important for pituitary development)

- Misregulated PIT-1 results in GH deficiency
- Low PIT-1 --> DWARFISM
- Children can be treated effectively w/ GH injections

22

Functions of the activating domain of regulatory proteins

HAT= histone acetyltransferase

Allows TF's to
1. bind other TF's
2. Help RNApol II form initiation complex
3. Recruits multisubunit complexes (HATS)

23

HATs HDACs

What is acetylated histone?

HAT- histone aminoactyltransferase
HDAC- histone deacetylases

HATs- Acetylation Cut Histones into smaller pieces and make DNA more accessible for Transcription

HDAC- puts them back together

ACETYLATED HISTONES= ACTIVE CHROMATIN

24

Transcriptional Repressors
4 modes of transcriptional repression:

inhibit tissue genes in inappropriate cell types

-binding motifs sames as activators (Znfinger, HtH,Leuzipper, HLH)

1. Competition (activator/repressor compete for same site)

2. Inhibition (repressor binds/inhibits by protein interaction)

3. Direct repression (repressor binds to repressor binding site and prevent TF's/RNApol function)

4. Indirect repression (recruits HDACs)

25

RNA interference (RNAi) or Post-transcriptional Gene Silencing (PTGS)

What is it? how big? What is its function in humans?

SHORT DOUBLE STRANDED RNA!!!
(21-23ntds)dsRNA

RNAi regulates gene expression in embryonic development to control the timing of development of various tissues

26

RNAi mechanism
4 steps:
siRNA = small interfering RNA

1. dsRNA cut into siRNA by DICER
2. siRNAs bind RISC (only one strand binds)
3. ssRNA hybridizes w/ mRNA target
4. mRNA cut or translation blocked

27

WET AMD
What is it characterized by?
How is it treated?
Outcome?

Wet Age-related macular degeneration
1. BV hyperplasia- leaks blood/fluid into eye (PAINFUL)

Treat w/ siRNA against VEGF (vasc endothel gf)
- inject into eye
-Inhibits bv growth, improved vision, extend time between injections

28

Reticulocytes inhibiting translation of Hb using eIF2

How does it work?

If no heme available, kinase phosphorylates eIF2
Blocks exchange of GTP for GDP (can't bind ribosome)
Hb Synthesis inhibited

eIF2 transfers initiator met-tRNA to Small ribosome
kinase activity blocks exchange of GDP to GTP
inhibited