MCM_Final_TBL10

Question 1

Expression Control: Prokaryotes

Answer 1

1. simple
2. REGULATED by transcription initiation
   
   • ​termination control mechanism
   • riboswitch mechanism
3. ​metabolites influence expression

Question 2

Expression Control: Eukaryotes

Answer 2

1. DNA packaging + 3D Gene location
2. transcription initiation + partial promoters
3. processing transcripts
4. stability of mRNA
5. initiation of translation

i.e very complex

Question 3

Prokaryotic Gene Expression: Operator DNA OFF

Question 4

Prokaryotic Gene Expression: Operator DNA ON

Answer 4

1. inducer molecule in cell
2. inducer forces repressor to fall off operon
3. sigma factor + RNA Pol can NOW BIND and transcribe
4. mRNA produced = more proteins

Question 5

Transcription in Prokaryotes

Answer 5

• trascription form DNA –> RNA, main site of control/ regulation
• each peptide is INDEPENDENTLY transcrilated
  
  • each have their own AUG start codon
• stop codon = Shine-Dalgarno sequence

Question 6

Lac Operon

Answer 6

• mainly used by bacteria to consume gluose sugar found in Lactose (galactose + glucose = lactose)
• when glucose levels are low, cAMP levels are higher, and more can bind to and activate CRP, allowing it to activate the operon.

If glucose is present in the cell, cAMP levels will be low, and only a small amount will be able to bind to the cAMP-regulatoed operon

Question 7

Glucose inhibits…

Answer 7

• adenylyl cyclase = NO cAMP

Question 8

Lac Operon: When Lactose NOT Present

Answer 8

• if no lactose, then no Lac ZYA digestion enzymes necessary to be made
• so if no lactose, repressor IS BOUND to operator
  
  • ​blocks RNA Pol from making LAC ZYA
  • defult

Question 9

Lac Operon: When Lactose PRESENT

Answer 9

• need the Lac ZYA enzymes to digest the lactose:

1. lactose molecule BIND repressor (which is still binded to the LAC OPERON)
2. repressor falls off operon
3. RNA Pol binds to LAC PROMOTER
   
   • mRNA encoding for Lac ZYA digestive enzymes produced

Question 10

Lac I

Answer 10

• has its own promoter
• gene that is upstream of the Lac operon
• encodes for the repressor of the Lac operon

Question 11

Promoter

Answer 11

• in bacteria, this is where RNA Polimerase binds to start transcription

Question 12

Operon

Answer 12

• found mostly in BACTERIA
• regulates the promoter region
  
  • when a repressor is bound, prevents transcription by blocking RNA Pol from binding to the promotor

Question 13

Protein: β- Galactosidase

Function?

Answer 13

• Lac Z
• breaks lactose into glucose and galactose

Question 14

Protein: Permease

Function?

Answer 14

• Lac Y
• transports lactose into cell
  
  • pumps lactose into cell using proton gradient

Question 15

Protein: Transacetylase

Answer 15

• Lac A
• acetylation of β-Galactosides: transfer of the acetyl group of acetyl-CoA to beta-galactosides
• may be important in detoxifying the bacteria by preventing acetylated galactosides from re-entering the cell.

Question 16

Riboswitch

Answer 16

• UNTRANSLATED 5’ region of bacteria
• contains:
  
  • ​Shine-Dalgarno Sequence
    
    • ​where ribosome binds
  • Regulatory elements that bind metabolite
    
    • ​metabolite bound = early transcription terminator
      
      • ​ feeback INHIBITION
      • ex) Vit B1 and B12

Question 17

Eukaryotic Expression Regulation: DNA Packaging + 3D Location

Answer 17

nuclues contains two chromatin types:

1. condensed heterochromatin: inactive as promoters are blocked
2. loose euchromatin: produce mRNA as promoters are accessible

EPIGENETIC Regulation:

• nucleosome = DNA wrapped around histone proteins
  
  • special histones will block the promoter region of DNA
• ACETYLATION of LYSINE in the histone tail = ACCESSIBILE promoter

Question 18

Epigentic Regulation: DNA Methylation

Answer 18

• CYTOSINE is methylated at the 5-position
  
  • in humans, occurs in context of CpG dinucleotides
• promoter region = high [] of CpG
  
  • when PROMOTER METHYLATED = NO transcription factor binging = NO protein
• TCA mutation and SAM mutations

Question 19

Angelman

Answer 19

• genes affected: Chromosome 15 (UBE3A)
• symptoms:
  
  • seizures
  • ataxia (inability to move, walk, balance)
  • intelectual disability
  • constant laughing
• biochemical pathway:
  
  • genetic imprinting = one gene is active (either the mom’s or dad’s); methylation of sperm or eggs is parent specific
  • cannot produce protein even though a correct copy is present

only the maternal copy of the UBE3A gene is active in the brain. Most cases of Angelman syndrome occur when part of the maternal copy is missing or damaged.

Question 20

Prader-Willi Syndromes

Answer 20

• genes effected: Chromosome 15 (OCA 2)
• biochemical pathway:
  
  • genetic imprinting = one gene is active (either the mom’s or dad’s); methylation of sperm or eggs is parent specific
  • cannot produce protein even though a correct copy is present
• Symptoms:
  
  1. ​​intellectual diability
  2. obesity
  3. constant hunger (hyperphagia)
  4. hypogonadism (low male hormone)
  5. almond shaped eyes
• methylation of the paternal essential gene forces a deletion

Question 21

Transcription Common Tools (1-3)

Answer 21

1. activator (transcription factor):
   
   • remove epigenetic marks
2. enhancers (transcription factor):
   
   • assists in BETTER recruitment of RNA Pol
3. Remodelers (transcription factor):
   
   • makes gene more acessible for transcription

Question 22

Partial Promoters

Answer 22

• recall p53 + promoter
• many Transcription Factors act on 1 gene
  
  • they will bind to the promoter with different affinities = different transcriptional effects
• TATA Binding Protein = 1 x mRNA (base lvl)
• P53 is also bound = 3 x mRNA produced
• Enhancher binds = 15 x mRNA produced

Question 23

Direct Hormone Signaling

Answer 23

1. Transcription Factor (TF) in cytosol and inhibited
2. Hornmone binds to TF = structure change
3. TF is freed and transports to nucleus
4. TF binds to promoter
5. transcription occurs

Question 24

Secondary Hormone Signaling

Answer 24

1. hormone binds G-Protein Coupling Receptor (GPCR)
2. secondary messeging system activated
3. activates bound transcription factor (the target protein) via phosphate modification

Question 25

Glucocorticoid Biochemistry

Answer 25

* **Glucocorticoid** = hormone *from **adrenal cortex*** to _reduce stress_ * mechanism * **cortisol** binds **to** a **GR dimer** (dimerization) * the **complex** will **bind** the **GRE promoter** to **enchance** (increase expression) or **repressor** (decrease) * outcome: * **replenishes blood sugar** * **reduces** the **immune** **response**

Question 26

Transcription Factors + **Cascading**

Answer 26

* **similar** mechanism as **secondary messaging** system of proteins * **one external signal** = activation of many transcription factors = **many genes transcribed**

Question 27

Stability of mRNA

Answer 27

* long half-life of mRNA = more cycles = more protein Review (TBL 8) 1. **mRNA cut at 3'- end** * polyA **tail** + 5' **cap** 2. **snRNA** select the **splicing** pattern 3. **mRNA exported OUT** of nucelus 4. mRNA has 5' & 3' **UNtranslated region** (_regulatory_ domains) 5. **ribosome** = small subunit (**40S)** + large subunit (**60S**) 6. if _mRNA in CLOSED LOOP_, **ribosome binds** 7. **polyA tail is cut off overtime**

Question 28

mRNA stability + **Iron**

Answer 28

1. **iron** consumed then **bound to transferrin** 2. *mRNA* for *transferrin receptor* (**TfR mRNA**) is ALWAYS present but not always made to protein * **HIGH iron** = 3'-UTR NOT protected (**unbound by IRP**) * cut by nuclease * no closed loop * **no TfR made** * **Low iron** = the Iron Recognition Element (**IRE**) is protected (**bound**) by Iron Recognition Protein (**IRP**) * **translation occurs** and continues until iron is high again

Question 29

RNA Editing & **Lipoproteins**

Answer 29

* lipids are carried in the bloodstream by **lipoproteins** (since lipids are not soluable) * Two Major Lipoproteins * **Chylomicron**: carries _dietary_ lipids from *intestine to circulation* * **VLDL**: carries endogenous lipid from *liver to blood* * *different markers for each on the **SAME gene*** * **​RNA is EDITED** to include an early **stop codon** depending on what is needed

Question 30

PCR and Mutation Probes What does it do? What does it require? How to apply it? Limitations?

Answer 30

* Polymerase Chain Reaction (PCR) * **AMPLIFIES target DNA sequences** present in small quantities * requires: 1. two **primers** (3' end and 5' end) 2. **dsDNA** 3. **Mg2**+ & **dNTPs** 4. _heat stable_ DNA polymerase (**Taq Polymerase**) * used for **genetic testing** * **allele specific probes** will diagnose genetic disorders * l**ocation and mutation determined quickly** * combined with *restriction* nuclease for analysis * LIMITATIONS: 1. **contamination** problems 2. **smaller** DNA made

Question 31

Southern Blot

Answer 31

* **labels DNA** with probe to **determine** gene **change**

Question 32

PCR Technique: Restriction Fragment Length Polymorphisms (**RFLP**)

Answer 32

gel test ex) **Sickle Cell** is caused by **mutation of β-globulin** * SC patient's DNA is PCR amplified and compared to normal controls * **restriction enzymes** *recognize* the **palindromic** PCR sequence in _NORMAL DNA_ _Outcomes:_ 1. **normal**: **palindrome** **retained**; PCR product SMALL 2. both alleles **mutated**: **palindrome** **lost**; PCR product LARGE 3. **heterozygote**: **both products** vizualized using *_Southern Blot_*

Question 33

Probe Screening

Answer 33

* Allele Specific Oligonucleotide **(ASO) probes** * **ASO probes are added with dye** * the presence of *mutation* of **codon 6 in β-globulin gene (**glutamate --\> valine) * **bs** peptide **= (+) sickle cell​** * **ba** peptide **= normal** * PCR DNA of β-globulin is _retained on the surface_

Question 34

PCR Technique 2: qPCR

Answer 34

* in **Prader-Willi syndrome** = one allele mutated and the other is **methylated** at **promoter** (preventing transcription) * how to detect methylation using mRNA 1. **isolate** **mRNA** via poly A tail 2. **convert** to **DNA** 3. **remove** **mRNA** 4. gene specific **PCR amplificaton** 5. **probe** (**NORTHERN** or **SYBRGREEN**) * outcomes: * _more green = more mRNA present_ (*normal)* * **Prader-Willi Syndrome = NO GREEN color**

Question 35

Northern Blot

Answer 35

* **labels RNA** to determine if gene is being actively expressed

Question 36

DNA Sequencing

Answer 36

* **Sanger** or Traditional DNA **sequencing** * used to determine mutation, insertion, deletion of DNA 1. **mitrochondrial profiling** (determine maternal lineage) 2. check for mutation 3. genetic disorders 4. whole genome * becoming *replaced by Next-Gen Sequencing* 1. **​​quantify all mRNA t**ranscripts 2. determine **ALL mutations** 3. **determine** inborn **errors** of **metabolism**

Question 37

Sanger Sequence Determination

Answer 37

* **dye-labeled nucleotide terminators and electrophoresis** are used * traditionally dideoxy * different colors for different bases

Question 38

Next-Gen Sequencing

Answer 38

* whole genome sequenced I**N HOURS** * scans entire genome for mutations * _categorizes population changes_ * 4 steps

Question 39

Next-Gen Sequencing: Step 1

Answer 39

***_Library Preparation_*** 1. **DNA** is **fragmented** 2. segments added to both sides (**barcoding**)

Question 40

Next-Gen Sequencing: Step 2

Answer 40

***_Cluster Generation_*** * fragment in physical **location**; _akin to PCR_ (called **polony)**

Question 41

Next-Gen Sequencing: Step 3

Answer 41

***_Sequencing_*** * **1 nucleotide at a time** using *reversible terminator* * _differs from machine method_

Question 42

Next-Gen Sequencing: Step 4

Answer 42

***_Data Analysis_*** * put sequence together

Question 43

Transcriptomic of Glucocorticoid

Answer 43

* how it works? 1. use **A-tail to collect patient mRNA** 2. use r**andom primer to amplify** 3. **count** the number of tines a given mRNA is **sequences** Ex) Effect of **Dexamethasone (**synthetic glucocorticoid) * used to *_treat overactive immune inflammation_* with many infications * more dexamethason = more mRNAs changed

Question 44

Molecular Cloning (Make Protein of Interest)

Answer 44

1. **isolate mRNA** 2. produce **cDNA** 3. **insert cDNA into plasmid** containing *_antibiotic resistance genes_* 4. insert _plasmid into bacteria with anibiotic media_ 5. **surviving bacteria have to express the protein of interest** 6. _isolate protein_

Question 45

mRNA Reduction via RNAi

Answer 45

**RNAi** is an *unnatural* technique used to **SILENCE** (reduce) **mRNA** (make **less protein**) * miRNA = part of translational regulation **_Drosha, RISC, Exportin normal enzymes_** 1. the **3'UTR is cut** or blocks ribosome 2. the mRNA has **no A-tail and is degraded (**HALTS TRANSLATION) \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ RNAi targeting receptor **leads to less hormone action** (i.e **reduction of glucocorticoid receptor mRNA**)

Question 46

Western Blotting

Answer 46

Method: 1. take **patient sample** and **seperate protein**s *via gel* 2. add **primary antibody that is diagnostic** for a disease 3. wash and **stain with secondary antibody** 4. **amount** of substrate is **determined**

Question 47

ELISA Assays

Answer 47

Method: 1. coat surface with sample (**antigens**) 2. block unoccupied sites 3. incubate with **primary antibody** against specific antigen 4. incubate with s**econdary antibody**-ezyme complex that binds primary antibody 5. add substrade 6. **formation of colored product indicates presence of specific antigen** **simpler and faster** than Western blotting, _WB can confirm results of ELISA_