Structure and expression of eukaryotic gene - Professor Latchman

Question 1

At what level does gene regulation primarily occur?

Answer 1

At the level of transcription

Question 2

What are untranslated regions of an mRNA

Answer 2

Segments of a gene that are transcribed into mRNA but are not translated into the final protein structure

Question 3

How many RNA polymerases are there in prokaryotes and eukaryotes?

Answer 3

1 in prokaryotes and 3 in eukaryotes

Question 4

Describe the 3 RNA polymerases in eukaryotes, what they transcribe and their sensitivity to alpha amantin

Answer 4

RNA polymerase I - rRNA (45S precursor of 28S, 18S and 5.8S), insensitive to alpha amanitin
RNA polymerase II - all protein coding genes, snRNAs U1,U2,U3, very sensitive to alpha amanitin
RNA polymerase III - tRNA, 5S rRNA, snRNA U6 etc, moderately sensitive to alpha amanitin

Question 5

Describe how researchers found out which RNA polymerase transcribed the protein coding genes

Answer 5

Alpha Amanitin is lethal to life as it inhibits RNA polymerase, different RNA polymerases have differing sensitivities to the compound. Researchers took a cell extract and gradually added small amounts of Alpha Amanitin and looked at what type of transcription was prevented. They knew that RNA polymerase II was the most sensitive so when the first type of transcription they lost was of all the protein coding genes they new that RNA polymerase II transcribes these genes.

Question 6

Why are the other RNA polymerases not regulated as much as RNA polymerase II?

Answer 6

Because RNA polymerase II is the RNA polymerase that transcribes protein coding genes, these genes need to be specific to each cell type. Other polymerases that transcribe rRNA or tRNA need to be present in all cells i the body and therefore are not regulated to the same degree.

Question 7

What is the most highly conserved promoter in eukaryotic protein coding genes? describe it

Answer 7

The TATA box, it is a region around the -30 region of a gene and it has the bases TATA in that order. It is found in the vast majority of protein coding genes, this tells us that it is very important but it does not tell us in what way it is important.

Question 8

Describe the Biochemical tests that were carried out on the TATA box and what conclusions were drawn from it

Answer 8

Researchers mutated the TATA box using site directed mutagenesis and they made the sequence read GCGC. They found that this mutation in the TATA box prevented transcription from occurring. Next they moved the wild-type TATA box down upstream form the -30 region to the -60 region. They found that transcription did occur but the start point of transcription had moved to the -30 position. They drew two conclusions, The TATA box not only controls the fact that transcription can occur, it also controls where transcription starts.

Question 9

Describe the process by which the TATA box leads to transcription

Answer 9

TFIID associated with TFIIA recognises the TATA box and TFIID binds to it. TFIIA facilitates the binding. TFIIB recognises the TFIID bound to the TATA box and binds to TFIID. RNA polymerase II is in complex with TFIIF and RNA polymerase recognises the complex and binds. TFIIH and TFIIE bind to the RNA polymerase. TFIIH is a kinase that phosphorylates the RNA polymerase and this allows transcription to start. The RNA polymerase in complex with TFIIF shoots off from the basal transcription complex and starts transcribing the gene. TFIIH phosphorylates the RNA polymerase at the C-terminal domain of the largest subunit at a serine molecule in a highly conserved region. TFIIE and TFIIH dissociate, TFIIA and TFIID are left on the TATA box, this allows the cascade to happen again without another DNA recognition event.

Question 10

What is the Basal transcription complex?

Answer 10

The full complex that is made during the cascade. This complex allows transcription to occur at a certain rate and this is known as the basal rate of transcription.

Question 11

What are upstream promoter sequences?

Answer 11

These are sequences that are upstream of the promoter that increase the rate of transcription by interacting with the basal transcription complex and the TATA box.
Two examples;
GC rich sequences
CAAT sequence - also termed the CAT box

Question 12

What binds to the GC rich sequence?

Answer 12

SP1, stands for species one

Question 13

What binds to the CAAT box?

Answer 13

CTF (CAT transcription factor)

Question 14

Are GC rich sequences and CAT boxes rare or common?

Answer 14

These upstream promoter sequences are very common, they are both found in the majority of protein coding genes. They are usually found on housekeeping genes that all cells need to transcribe as all cells create SP1 and CTF, so by having a GC rich sequence or a CAT box these two can bind and increase the rate of transcription. A gene that is only transcribed in a specific cell type would not have these sequences as the cell will probably make SP1 and CTF so if the gene had these sequences it will be turned on in all the cells that don’t need it.

Question 15

How do SV40 virus genes get transcribed in Human cells?

Answer 15

They have promoter sequences that are recognised by the host genome. SV40 has a TATA box and a GC rich sequence that SP1 can bind to

Question 16

Define an enhancer

Answer 16

An enhancer is defined on the basis of its ability to activate transcription from a promoter independently of its distance, orientation or position relative to the promoter.

Question 17

How do enhancers contribute to gene control?

Answer 17

The vast majority of enhancers that we have are cell or tissue specific and therefore will only increase the rate of transcription of certain genes in certain cell types.

Question 18

How does an enhancer work?

Answer 18

It is still not fully understood how an enhancer works but there is a widely accepted theory. The enhancer unwinds chromatin structures near specific genes so that transcription factors can bind to the DNA and increase the rate of transcription. The enhancer unwinds the chromatin in both directions and this is why it doesn’t matter what position or orientation the enhancer is.

Question 19

Describe the addition of a 5’ cap to an mRNA

Answer 19

The addition of a 5’ cap is the first post-transcriptional modification to occur, an extra G residue is added. This G residue is not in the DNA template encoded by a C residue it is added after transcription. The Guanine that is added has an extra methyl on its 7th carbon so its 7-Methyl-Guanine. The bond between the extra guanine and the first copied base is a 5’ to 5’ bond. The free 5’ end of the carbon of the last base is linked to the 5’ carbon on the guanine sugar and there are three phosphates in between.

Question 20

How are mRNAs directed to ribosomes in prokaryotes?

Answer 20

The ribosome recognises a specific sequence on the prokaryotic mRNA called a shine-dalgarno sequence. Prokaryotic mRNA is polycistronic and therefore one mRNA can have many ribosomes on it transcribing many different types of proteins.

Question 21

Describe how mRNAs are directed to ribosomes in Eukaryotes

Answer 21

Eukaryotes are monocistronic, one mRNA codes for one protein in one ORF, with one start and one stop codon. The ribosome recognises the 5’ cap and this is how it binds to the mRNA. The ribosome then scans along the mRNA sequence until it finds the start codon.

Question 22

Why is the 5’ cap needed?

Answer 22

The vast majority of RNA in eukaryotes is rRNA, the 5’ cap distinguishes mRNA from rRNA so that the ribosomes are not pointlessly translating rRNA all the time.

Question 23

Describe how the polio virus RNA is translated in host cells despite not having a 5’ cap

Answer 23

In the viral particle there is a cap binding protein that binds to the end of the viral RNA and mimics the 5’ cap of eukaryotic mRNA. This viral protein also cuts off the 5’ cap of host mRNA and eliminates the competition.

Question 24

Describe the mechanism of polyadenylation

Answer 24

There is a sequence at the 3’ end of the RNA that is slightly upstream of the polyA site that has a highly conserved region of AAUAAA. If these region is mutated then polyadenylation will not occur, this this region is moved then polyadenylation is also moved. This specific sequence is recognised by a CPSF protein, this protein then binds to this region. Another sequence downstream of the polyA site, is recognised by another protein, CstF and this protein binds to this sequence that is very GU rich. Both of the bound proteins CPSF and CstF then interact with each other, this causes the RNA to fold back on itself. A endonucleolytic cleavage then occurs between the two sequences. Two RNAs are then produced the GU rich RNA bound to CstF is degraded, the other RNA with the AAUAAA sequence bound to CPSF is more stable, the enzyme polyA polymerase then adds the polyA tail to the stable RNA, the process of polyA only occurs for protein coding RNA.

Question 25

How is having a polyA tail on an mRNA useful in a lab?

Answer 25

Can be used to isolate mRNA from other RNA in a cell by hybridising to a column rich in T residues and then eluting by changing the salt concentration.

Question 26

Why is having a polyA tail on an mRNA useful in a cell?

Answer 26

The polyA tail protects the mRNA from degradation by exonucleases in the cytoplasm. The tail acts as a buffer for the exonuclease, gives it something to degrade without degrading the protein coding sequence. If an mRNA didn’t have a polyA tail then it would be degraded very quickly by the exonucleases in the cytoplasm.

Question 27

Why don’t Histone RNAs have polyA tails

Answer 27

They stabilise their mRNA through forming a loop structure at the 3’ end. This gives the cell more control over the Histone proteins. During S phase when Histones need to double, this loop prevents exonuclease degradation and allows more Histones to be translated. Other times during the cell cycle where Histones are not needed as much, the loop does not form and the Histone mRNA are broken down.

Question 28

What are the conserved regions of the exon/intron boundaries?

Answer 28

The first two bases of an intron are normally GU and the last two bases are normally AG. Obviously there is more regulation then just these two dimers as these would occur on average 1/16 times in the genome. If you mutate these areas then you prevent splicing from occurring.

Question 29

What is the 5’ splice site

Answer 29

Exon going to intron

Question 30

What is the 3’ splice site

Answer 30

Intron going to exon

Question 31

Describe how researchers found out that splicing occurs in the nucleus.

Answer 31

The mRNA for the globin gene is 9S in the cytoplasm, the nuclear mRNA of globin is 15S. The researchers then hybridised both of these mRNAs to the original DNA of the gene, the 15S hybridised perfectly, the 9S hybridised but the DNA had to form loops in order for the sequence to match, this proved that splicing occurred in the nucleus.