Gene expression -Miska

Question 1

What provides the energy for RNA synthesis?

Answer 1

Hydrolysis of ribonucleotide triphosphate - the terminal phosphoanhydride bond of nucleotide triphosphate is hydrolysed (ATP)

Question 2

In which direction does RNA synthesis progress and why is RNA single stranded?

Does transcription require a primer?

Answer 2

5’ to 3’ as in replication ; the template strand is read 3’ to 5’ to make RNA 5’ to 3’ equivalent to the coding strand

Because only one of the DNA strands is transcribed, either strand can be the coding strand

No primer is required unlike in replication

Question 3

What is the sense strand of DNA?

Answer 3

The coding strand, the antisense strand is the non coding strand of DNA or template strand.

The coding stand is identical to the RNA transcript made

Question 4

What is the +1 nucleotide in the coding strand?

Answer 4

The transcriptional start site representing the first nucleotide that will be in the transcribed RNA

To the left of the +1 site (upstream/towards 5’) is the -1 nucleotide
There is no 0 nucleotide
Further downstream will have positive numbers +2 etc

Question 5

Which direction is upstream?

Answer 5

The 5’ direction

Question 6

What is the prokaryotic RNA polymerase made up of?

Answer 6

2 x alpha
1 x beta
1 x beta’
1 x sigma (dissociates and binds to multi-protein complex to form the holoenzyme)
The sigma conveys specificity; which genes to transcribe

Question 7

How many RNA polymerases do eukaryotes have?

What do they do?

Which is most highly regulated?

Answer 7

Three
RNA Pol I - rRNA precursors
RNA Pol II - mRNAs for proteins, transcribed the largest number of genes out of the three
RNA Pol III - ssRNAs like tRNA and snRNA

Which are similar structurally but catalyse transcription of different subsets of genes
Plus those in the mitochondria and chloroplasts of bacterial origin

Eukaryotic polymerases have more sub unit

RNA pol II is most highly regulated at initiation, elongation, termination, splicing, polyadenylation etc

Question 8

What is a cis element?

Answer 8

A region of non-coding DNA regulating the transcription of nearby genes eg promoter

Question 9

How can you identify trans-acting factors required for transcription initiation?

Answer 9

Transcription assay:
Take extract, DNA template and radioactively labelled ribonucleotides a and incubate at 30degrees C for half hour.

Transcription complexes are assembled, detect products by auto radiography b

Question 10

Where is TATA box commonly found?

What is it?

What do prokaryotes have instead of the TATA box as their promoter?

Answer 10

-25 to -35 of start site (on the coding strand)

8bp AT rich element, promoter in eukaryotes for Pol II
The promoter increases in complexity with the complexity of the organism

The -10 region and the -35 region

Question 11

What can aberrant regulation of gene expression cause?

Answer 11

Disease eg retinoblastoma , a heritable cancer of the eye caused by a mutation in the retinoblastoma (Rb) gene

Question 12

Where are the different eukaryotic RNA polymerases active?

Answer 12

Pol I - nucleolus (rRNA)
Pol II - nucleoplasm (mRNA)
Pol III - nucleoplasm (tRNA)

Question 13

What are the general transcription factors (basal factors) which aid Pol I, II and III?

What do they do?

Answer 13

TFIIB, TFIID TFIIA, TFIIF, TFIIE, TFIIH
They tend to be specific for Pol I, II or III
They control the rate of assembly or activity of the polymerases and assembly of he pre-initiation complex

Question 14

What is TFIID made up of?

Where does TBP bind and what does it do?

Answer 14

TFIID is a general transcription factor made up of the TATA binding protein TBP plus TBP associated factors (TAFs)

TBP with the rest of the proteins making up TFIID form the general transcription factor which in turn makes up part of the RNA Pol II complex

TBP binds to the TATA box in the minor groove helping to position the polymerase over the DNA and locate the start site (though not all RNA pol II promoter sites have TATA boxes)

TFIID nucleates the assembly of other general TFs

Question 15

What does TBP do to the DNA?

It is conserved?

Answer 15

Forms a molecular saddle bending the DNA

It is highly conserved suggesting it evolved before split of pol I II and III

Question 16

What do TAFs do?

Answer 16

They may bind to promoter sequences in cases where the promoter doesn’t contain a TATA box (so they do the role of TBP)

They may interact with activators or co-activators

They may bring about specific programmes of gene expression

Question 17

What does TFIIA do?

What does TFIIB do?

Answer 17

Binds and stabilises interaction of TFIID with DNA

Bridges between RNA pol II and TFIID
Also helps to locate transcription start site by binding to the Bre element upstream of the TATA box

Question 18

What does TFIIH do?

Answer 18

Uses energy from ATP hydrolysis to act as a helicase, for DNA nucleotide excision repair and promoter opening.

Also acts as kinase by phosphorylation RNA pol II CTD (C terminal repeat region on the largest subunit of the polymerase) which helps to link together transcription and RNA splicing, polyadenylation and mRNA export

The repeat in humans in 52 repeats of the consensus sequence YSPTSPS, this is quite conserved across the pol II of eukaryotes but there is no CDT equivalent in pol I or III

Question 19

What is a zinc finger?

What other structural motifs are there required in proteins to bind to DNA?

Answer 19

A structural motif characteristic of DNA binding proteins like TFs. The finger interacts with five base pairs in the nucleic acid molecule

Leucine zipper
Helix-turn-helix
Helix-loop-helix

Usually interact with double stranded DNA in the major groove

No factors known to interact with RNA?

Question 20

Why doesn’t RNA has a major groove?

Answer 20

It is single stranded

Question 21

How does the lac operon work?

Answer 21

Lac Z - beta galactosidase (cleaves lactose to glucose and galactose)
Lac Y - lactose permease (lactose uptake)
Lac A - lactose transacetylase

Further upstream of lac operon
Lac I - inhibitor protein

When no lactose lac I is expressed and inhibitor protein prevents RNA pol from binding to promoter

When lactose present allolactose (inducer molecule) binds to inhibitor which dissociates form promoter
Low glucose levels mean more cAMP to bind to CAP (activator protein)
CAP with cAMP binds upstream of promoter and promotes RNA pol binding

Question 22

What is the difference between TFs that bind directly to DNA and those that are indirectly recruited?

Answer 22

Those that bond indirectly are co-activators and co-repressors e.g TFIID and they lack DNA binding domains (the motifs like leucine zippers)

Question 23

How can transcription be regulated?

Answer 23

TF - eg RNA pol doesn’t bind directly to promoter, requires proteins sequence specific for promoter that also associate with RNA pol. Eg sigma factor in prokaryotes and GTF in eukaryotes

(Unsure about co-activators and co-repressors - I think they are associated with TFs so play a role in regulation but because they lack DNA binding domains they aren’t TFs

Regulate the regulators:
Modulate the levels of a factor 
Modulate their intrinsic activity - eg ligand has to bind before the TF can be active; the steroid hormone receptors act this way, binding of steroid hormone to the receptor in cytoplasm causes conformational changes revealing NLS and DNA binding region so receptor-hormone complex can act as TF 
Control by phosphorylation 
- eg p53

Transcriptional cascades - TFs can regulate TFs which regulate TFs etc in transcriptional cascades. The TFs at the top are master regulators

Organisation of subsets of genes related to similar functions into operons e.g lac operon or tryptophan switch (these require TFs) also called transcriptional switches

Changes in chromatin structure (torsten’s lectures) TFs may modulate changes in this environment

In bacteria transcription can also be regulated by attenuation as transcription and translation take place at the same time in prokaryotes. Secondary structures called attenuators form which regulate termination; attenuation results in termination of transcription.

Question 24

What is a mediator complex?

Answer 24

A co-activator bringing together GTFs RNAPII and TFs (like TFIID). Polypeptide complex that is conserved from year to higher eukaryotes. Something about SAGA complex

There are Multiple transcription factors per promoter

Question 25

How does p53 work?

Answer 25

p53 is normally bound by mdm2 (an E3 ubiquitin ligase) causing it to be exported to cytoplasm and destroyed

DNA damage (eg sunburn) causes p53 phosphorylation
Mdm2 dissociates and p53 is stabilised
Levels of p53 increase and it acts as a positive TF by binding and activating transcription of genes
Target genes regulate apoptosis and cell cycle control

Question 26

If histone acetyl transferases (HATs) open up chromatin (eg H3K9 acetylation) allowing access to DNA for TFs what can they be called?

Answer 26

HATs are co-activators and histone deacetylases (HDACs) are co-repressors

Question 27

How can chromatin be remodelled?

Answer 27

Histone acetylation by HATs, removes positive charge and opens up chromatin (HATs = co activators)
HDACs remove acetylation repressive to transcription (co-repressors)

Methylation - H3K9me attract HP1, attracts proteins to compact DNA
Methylation preserves positive charge
Enzymes = histone methyltransferases
Methylation compacts to transcriptionally inactive state

Chromatin remodelling complexes - use energy from ATP hydrolysis to mobilise nucleosomes, facilitates ability to slide or be pushed around

Question 28

How are HDACs used in thyroid hormone complexes and hence transcriptional regulation?

Answer 28

Absence of ligand causes the thyroid hormone receptor to bind to HDAC ‘SMRT’ repressing transcription

Ligand binding causes SMRT to dissociate, recruiting HAT to activate transcription

Question 29

What is splicing?

Answer 29

Removal of introns from mRNA after gene has been transcribed, differential splicing in same gene creates different proteins (100 000 proteins in humans)

Question 30

What marks the transcriptional start site? (+1 site)

Answer 30

Cozaki fragments either side of +1 nucleotide

Question 31

What is ChIP and ChIP seq?

Answer 31

Chromatin immunoprecipitation (ChIPseq is the same followed by DNA sequencing) add formaldehyde to cross link DNA to DNA and the associated proteins, fixing genome, Cut DNA, , use antibody specific to TF/DNA binding protein you want to find which is bound to its DNA sequence, once found isolate the TF and associated DNA, remove TFs and other proteins by digestion to leave associated DNA, amplify with PCR and sequence to give DNA that the TF you were looking for binds to

Question 32

What has ChIP taught us?

How does it differ from HiC

Answer 32

Where proteins associate to DNA enabling us to understand chromatin landscape in cells

HiC tells you where DNA associates to DNA, ChIP tells you where proteins associate to DNA

Question 33

Other than ChIP how else can we find out where proteins bind to DNA?

Answer 33

DNA foot printing

Amplify DNA fragment with Phosphate32 (radioactive) primers
Split sample between two test tubes
Add DNA binding proteins to one tube
Add DNAse (deoxyribonucleases) to both tubes to digest DNA (limited amount so as not to completely digest)
Proteins in the tube they were added to will protect where they are bound from digestion
Run on gel
Compare the fragments in the two tubes to see where proteins were bound

Question 34

What are enhancers and where are they?

What mediates looping?

What regulates looping?

Answer 34

Cis elements involved in regulation of transcription that can be far far away from the transcription units they regulate

DNA is thought to loop back bringing them closer to the promoter

Higher order protein structures like enhanceosomes (highly intricate and precise, each bp matters) or billboards (loosely collaborating elements)

Question 35

What is an insulator?

Answer 35

A factor that blocks the interaction between enhancers and promoters to prevent expression

Question 36

What is polyadenylation?

Answer 36

The addition of a poly(A) tail to mRNA. This tail consists of only adenine bases and is part of the process that produces mature mRNA for translation