Molecular Cell Biology Midterm 2 full review (Lectures 9-10)

Question 1

Given that all our cells have the same exact genome, what leads to their differences?

Answer 1

Gene expression

Question 2

What are the different levels at which gene expression can be regulated?

Answer 2

1-Transcriptional
2- RNA processing
3- RNA transport and localization
4- Translational
5- mRNA degradation
6- Protein activity

Question 3

What is RNA seq?

Answer 3

Sequencing all RNA in the cell (transcriptome). At each position along the genome, the height of the coloured trace is proportional to the number of sequence reads that match the genome sequence at that point.

Question 4

How is RNA seq done?

Answer 4

Take cell lines, grind them up, extract mRNA. This is done using poly-T beads which are complementary to poly-A tail. Purify, then add reverse transcriptase to obtain cDNA. Give this to a sequencing company, then get readout.

Question 5

Why are the intron sequences present on the readout?

Answer 5

They are present at lower loves and reflect pre-mRNA molecules that have not yet been spliced plus intron sequences that have been spliced but are not yet degraded.

Question 6

What do transcription regulators bind to?

Answer 6

Cis-regulatory sequences by means of the major and minor grooves of DNA.

Question 7

Which groove is used most often?

Answer 7

The major groove.

Question 8

How do the DNA sequences affect transcription regulator specificity?

Answer 8

A protein can only interact with a certain base pair due to its hydrogen bond pattern.

Question 9

What can a transcription regulator recognize on the DNA?

Answer 9

Either nucleotides or the backbone. The interaction with the backbone is not strong enough for binding alone.

Question 10

What are the two general ways in which DNA binding proteins interact with DNA?

Answer 10

1 - Contact with specific bases mainly through major groove, also via minor groove.
a) Each base pair can form H-bonds (or hydrophobic interactions) with DNA binding proteins
2 - non-specific contacts with phosphates of DNA backbone

Question 11

What are the different structural binding motifs that can read DNA sequences?

Answer 11

Helix turn helix, Homeodomain proteins, Leucine zipper proteins, Zinc finger proteins,

Question 12

Most transcription regulators will not activate transcription alone. How then do they increased their affinity and specificity for DNA?

Answer 12

Through dimerization. Most transcription factors form homodimers or heterodimers. Generally, a typical Treg will recognize 6-8 nucleotides, which is not very specific. Interaction to form a dimer increases both affinity and specificity.

Question 13

What is the difference between transcription regulators that have strong affinity for each other and those that display cooperative binding? Draw the binding curves to support your answer.

Answer 13

For those that have strong affinity, they act as monomers and there is no time in the cell where they are not bound, with an increase in concentration of these, there is an exponential increase in occupancy of DNA, until saturation.
For those displaying cooperative binding, there is not a strong interaction between the subunits. DNA binding of the subunits strengthens dimerization. This results in an all-or-none occupance of the DNA. The binding curve is sigmoidal.

Question 14

Where do transcription regulators bind preferentially?

Answer 14

Bind to DNA outside nucleosomes preferentially.

Question 15

What are the two effects that the histone core has on Treg binding?

Answer 15

Either blocks the access or forces a specific bending of the DNA that may not be compatible with the TF binding.

Question 16

What can expose the regulatory sequences for the Tregs?

Answer 16

Breathing of nucleosomes which occasionally exposes regulatory sequences.

Question 17

How does the nucleosome structure promote the cooperative binding of TRegs?

Answer 17

One Treg can destabilize the nucleosome, facilitating binding of another. (mark this as five he said to skip)

Question 18

What does a ekaryotic gene control region typically consist of?

Answer 18

One promoter region and many cis-regulatory sequences which can be bound by both Transcriptional activators and repressors (TAs and Treps). These can either promote, or inhibit RNA polymerase binding to the promoter.

Question 19

How do co-activators promote RNA polymerase binding to the promoter?

Answer 19

Either directly, by also binding to RNA polymerase to help to stabilize or recruit RNA polymerase binding to the promoter. or,
Indirectly, where co-activators bind to histone remodeling proteins, histone modifying enzymes which cause displacement or modification of histones, opening up the promoter to allow RNA polymerase binding.

Question 20

What is the mediator complex?

Answer 20

Has a lot of factors and subunits, involved in the activation of many genes, interacts with activators and core machinery.

Question 21

What are the different ways that proteins that bind to transcription activators remodel chromatin?

Answer 21

Nucleosome sliding, removal and replacement.

Question 22

What are the histone modifications?

Answer 22

histone acetylation and methylation.

Question 23

What are HATs?

Answer 23

Histone acetyl transferases which acetylate histones and promote RNA polymerase binding.

Question 24

What are histone methyl transferases?

Answer 24

Methylate histones which promotes closed chromatin configuration to prevent RNA polymerase binding.

Question 25

What are deacetylases and demethylases and what are their effects on transcription?

Answer 25

Deacetelysases remove acetyl groups on histones and reduce RNA polymerase binding. Histone demethylases remove methylases from chromain and promote RNA pol binding.

Question 26

What is transcription activator synergism?

Answer 26

Transcription activators often barely have an effect on transcription when binding independently but when both bind, their synergistic effect is to greatly increase transcription.

Question 27

What are the different methods in which transcription repression can occur?

Answer 27

By competitive DNA binding.
By masking the effect of an activator.
By direct interaction with transcription factors.
Recruitment of chromatin remodeling complexes.
Recruitment of histone deacetylases.
Recruitment of histone methyl transferases.

Question 28

What are insulator seqences?

Answer 28

Insulate a gene from the effects of a different cis regulatory sequence through looping of the DNA sequence by interacting proteins.

Question 29

Describe the experiment with the TE white gene with the scs flanking sequences.

Answer 29

Take transgene that encodes white protein. This transgene lacks cis-regulatory elements and therefore has a low level of transcription on its own. Flank this transgene with scs sequences. Control transgene contained same gene but with random sequences flanking it.
The one with the scs sequences will not be expressed regardless of where it inserts in genome. The other transgene will be highly expresses if near a strong enhancer region. Causes red eyes.

Question 30

Does a single scs sequence insulate the gene?

Answer 30

No, need scs and scs’ to flank gene.

Question 31

What proteins associate with each sequence?

Answer 31

Zw5 binds to scs sequence.

BEAF binds to scs’ sequence.

Question 32

How did they determine that these proteins interact?

Answer 32

Did a GST pulldown with Zw5-GST fusion protein that had bacterially purified BEAF associated with it.

Question 33

Describe Bcd. Where is it deposited? What does it form? What kind of transcription regulator is it? What type of gene is it?

Answer 33

Bcd mRNA is deposited in egg during oogenesis by mother.
Bcd protein forms a gradient from anterior towards middle of egg.
Bcd is a homeodomain transcription factor that promotes transcription of genes required for anterior fates. (can also bind RNA and regulate translation). Bcd is genetically required in the mother-maternal effect gene.
It’s targets are otd, hb (and others).

Question 34

What is the difference between the affinities and binding sites of Otd and Hb.

Answer 34

Otd has 3 weak sites and lower affinity for Bcd, therefore needs to be present in higher concentration of Bcd to be expressed.
Hb has 3 weak sites and 3 strong sites and can be expressed in lower concentrations of Bcd.

Question 35

What is the Bcd protein gradient?

Answer 35

Decreasing concentration from the anterior towards the posterior of the embryo.

Question 36

What are the target genes of Bcd (other than Otd)?

Answer 36

Hb, Giant and Kruppel.

Question 37

What does Hb repress?

Answer 37

Kruppel transription.

Question 38

Where is Kruppel expressed?

Answer 38

Where there is low concentration of Hb and enough Bcd for induced expression. Kruppel has high affinity binding sites for Bcd.

Question 39

What was the experiment to determine the different even skipped regulatory sequences?

Answer 39

Excise regulatory sequence from normal DNA, upstream of Eve gene TATA box. Then insert this in reporter gene, LacZ that makes B-galactosidase, upstream of TATA box. B-galactosidase is expressed in single stripe in embryo. (X-gal to visualize it)

Question 40

How would you determine which proteins interact with the regulator sequence of Eve 2?

Answer 40

Chromatin IP.
Cross link proteins to DNA regulatory sequence using formaldehyde.
Lyse cells.
Break DNA into small fragments.
Many other proteins will be interacting.
Pass the mix over beads with antibody against transcription regulator of interest.
Spin. These will be in pellet.
Reverse cross link.
Elute protein and sequence it.
Number of sequence reads indicates frequency of protein occupancy-relates to affinity (and protein concentration).

Question 41

What are the combinatorial controls for the Drosophila Eve gene?

Answer 41

Has binding sites for Bcd, Hb, Giant and Kruppel.
In order for Eve 2 to be transcribed, Bcd and Hb must be bound, these have a synergistic effect on RNA polymerase recruitment.
Giant and Kruppel concentrations must be low, since they are thought to repress transcription.