Week 3

Question 1

in vivo

Answer 1

in the living cell/organism

Question 2

in vitro

Answer 2

in the test tube

Question 3

in silico

Answer 3

‘bioinformatics’ i.e. in the computer

Question 4

Abreviation for the promoter of the lac operon

Answer 4

usually you’ll see ‘P-lac’ indicating the ‘lac promoter’

Question 5

Lac I-q

Answer 5

this is a mutation which overexpresses the LacI protein, resulting in much more stringent control of the genes under its regulation

Question 6

meaning of the brackets around [cAMP]

Answer 6

square brackets mean ‘concentration’ – here, ‘concentration of cAMP’

Question 7

7 levels of regulation

Answer 7

• Transcriptional Regulation
• mRNA processing
• mRNA transport -

Sequestration -

Translation -

Degradation -

Protein function (Post-translational modifications)

Question 8

mRNA

Answer 8

code for proteins. Only 3-5% of total RNA in a mammalian cell

Question 9

tRNA

Answer 9

central to protein synthesis as adaptors between mRNA and amino acids

Question 10

Southern blotting

Answer 10

measures DNA. Uses DNA/RNA as a probe

Question 11

Northern blotting

Answer 11

measures RNA. Uses DNA/RNA as a probe

Question 12

Which one is a better probe? RNA or DNA?

Answer 12

DNA. It is more stable than single stranded RNA

Question 13

what does Western blotting measure? what is the probe?

Answer 13

measures Protein. Uses antibodies as a probe

Question 14

Agar is derive from? Used for?

Answer 14

a polysaccharide derived from seaweed, used in cooking (like gelatin except gelatin is protein-based) Few bacteria have agarase to digest agar so its good for culturing bacteria. Big pieces of DNA. 10-100 bps

Question 15

Acrylamide is used for?

Answer 15

can be cross linked to form polyacrylamide gel. Used for smaller isolations. 1 bp

Question 16

How does Southern/Northern blotting hybridization work

Answer 16

nucleic acids put in a gel are negatively charged. An electric field is applied and the acids will migrate toward the positive pole.

Question 17

Transcription regulators

Answer 17

Cis: intramolecular/ from the same molecule

Trans: intermolecular/from a different molecule

Question 18

Sigma factors

Answer 18

Activate a group of genes at once

Question 19

RNA polymerase characteristics

Answer 19

 Error rate ~10-4 (but remember: degeneracy of genetic code etc.)

 Elongation rate ~20-50 bases/sec (bases sec-1) (~1/10th of DNA rate)

Core enzyme & Holoenzyme

 So, sigma allows RNApol to slide along DNA ~easily until it finds a promoter, then binds tightly, starts transcription, releases sigma

 Dissociation constant: the concentration that allow 50% binding / 50% unbound

Question 20

Core enzyme vs Holoenzyme

Answer 20

Core enzyme (without σ) does not specifically bind promoters, but rather dsDNA very tightly

KD ≈ 5 x 10-12M t1/2 ≈ 60 minutes • Half-life is 60 minutes

Holoenzyme binds non-promoter DNA more loosely

KD ≈ 10-7M t1/2 > 1 sec • Lower KD= higher binding

Question 21

what is Lactose

Answer 21

Disaccharide: glucose and galactose. Enzyme lactase is necessary to clip lactose into 2 monosaccharides

Question 22

Lac operon proteins

Answer 22

Lac I: repressor protein

Lac Z: b-galactoside (cuts lactose)

Lac Y: Lactose permease ( membrane protein)

Question 23

Binding of lac repressor

Answer 23

Tetrameric lac repressor interacts simultaneously with two sites near the lac promoter

DNA loop forms

RNA pol can still bind to the promoter

Question 24

lac operon regulation

Answer 24

Lac I binding to DNA is NOT covalent, thus its not on the molecule 100% of the time bc binding is based on KD. Thus it is “leaky” and there is always a low level of expression of the operon. Which means there is always a tiny bit of each protein around

Question 25

Define Induction. What causes it?

Answer 25

Turn on (activate) gene expression, typically due to some change in the environment detected by some metabolite

Question 26

Define Repression, what causes it?

Answer 26

Turn off (inactivate) gene expression, typically due to some change in the environment detected by some metabolite

Typically, this is due to presence of the SUBSTRATE of a pathway (activation) or too much PRODUCT (inactivation)

Question 27

DNA binding proteins bind to…? What are their functions?

Answer 27

Three main classes

Typically bind in major groove, at palindromic sequences using alpha helices

Many use Zinc ions (Zn++) as cofactors

These are so critical because they regulate gene expression

Question 28

Gratuitous/exogenous inducers/repressors function

Answer 28

Can be added to cells. Turn on or turn off expression but are unaffected by the proteins made

Question 29

Mechanism for Gene Transcription in Eukaryotes

Answer 29

Enhancers

• not fixed in location
• bidirectional
• they even work downstream of gene
• ‘promiscuous’ as they work with almost any gene, but they do require specific transcription factors

Response Elements

• Allow for more specific responses (similar role to s factors in bacteria). E.g. HRE (heatshock element), which is bound by a specific heat shock transcription factor (HSTF)
• MRE, GRE, etc

Question 30

How are genes transcribed in eukaryotes?

Answer 30

RNA polymerases I, II and III transcribe rRNA, mRNA and tRNA genes, respectively

RNA Pol III transcribes a few other RNAs as well

All 3 are big, multimeric proteins (500-700 kD)

Question 31

Transcription in eukaryotes

Answer 31

More complex than prokaryotes, promoters, enhancers, response elements

Many more Transcription Factors (TFs)

Nucleosome structure (DNA wrapped around histones) requires additional enzymes for de/acetylation of histones to unwind DNA

No operons

NB – there’s another big complex of 18 proteins called Mediatorinvolved