Regulating Gene expression

Question 1

How many genes are in the cell?

Answer 1

There are 19-23,000 genes, and about 20,000 of these are expressed.

Question 2

What are common structural proteins and enzymes?

Answer 2

Different cell types express the same proteins.
Histones, RNA polymerases, B-actin, tubulin, glycolytic enzymes.
Encoded by housekeeping genes.

Question 3

What genes are in the cell?

Answer 3

Most individual cells express 10,000 - 15,000 genes.
Many are housekeeping proteins required to keep the cell alive and perform common functions.
Some are specialised proteins not essential for viability but for the specific function of the cell.

Question 4

What rate are genes expressed at?

Answer 4

Abundant - 1000-10,000 mRNAs per cell.
Scarce 10-100 mRNAs per cell - the majority.

Question 5

What are the categories of gene expression?

Answer 5

Constitutively expressed genes - always on.
Conditionally expressed - inducible.

Question 6

How can gene expression be controlled?

Answer 6

Chromatin marks whether genes are switched on permanently.
Histones can be methylated or acylated.
RNA polymerase can be switched on or off by different factors.

Question 7

How is transcription controlled?

Answer 7

A high level of RNA usually produces a high level of transcription.
But this assumes equal stability.
There may be lots of RNA but a slower rate of transcription so little protein present.

Question 8

How is the mRNA population regulated?

Answer 8

Which genes are transcribed - active vs repressed.
The rate at which genes are transcribed.
The transcriptional start site used.

Question 9

What can splicing be used for?

Answer 9

Can be used to increase the number of different proteins present in the cell.
Most exons in multi-exon genes can have alternative splicing events where different combinations of exons can be assembled as you splice out the introns.

Question 10

When does splicing occur?

Answer 10

At the same time as the process of transcription capping polyadenylation.

Question 11

Where are splicing factors?

Answer 11

Splicing factors are on carboxy-terminus of the RNA polymerase complex and as synthesised RNA is released, it is then available to interact with the splicing factors. This is a co-transcriptional process.

Question 12

What is the size of introns and exons?

Answer 12

The majority of genes are introns which are 10,000-100,000 bp long.
<5% of genes contain a single exon, 80% of which are <200bp long.

Question 13

How can splicing be seen?

Answer 13

Exons are very small so hard to see where they are.
PCR can be used to amplify the DNA to work it out.
Primers are placed where the exonic sequences are thought to be and then PCR is run to check.

Question 14

What is the function of introns?

Answer 14

Important in molecular evolution for exon shuffling.
Allows for alternative splicing - can produce multiple proteins from the same gene.

Question 15

What do introns look like?

Answer 15

Usually AG GU on 5’ end, and AG G on 3’ end which indicates it is an intron sequence.
The branch point nucleotide in the middle is involved in initial splicing on intron sequence.
The intron is then removed and degraded.

Question 16

What are invariant sequences?

Answer 16

GU and AG of intron are invariant but need additional sequences associated to make them functional.

Question 17

What is the splicesome?

Answer 17

The mRNA splicing machinery.
Contains snRNPs.

Question 18

What are snRNPs in the splicesome?

Answer 18

Small nuclear ribonucleoprotein complexes.
Contain snRNAs, which are U1, U2, U4, U5 and U6.
Proteins are associated with snRNA so they have specificity and can recognise RNA intronic and exonic boundaries and allows proteins to associate with them.

Question 19

What are RNBPs?

Answer 19

RNA Binding Proteins recognise splicing enhancers and repressors in RNA.
e.g. heterogenous nucleoribous proteins (hnRNPs), Serine rich splicing factors. (SRFs).

Question 20

What is cellular context?

Answer 20

Different cells will carry out processes differently:
Signal is produced so RNA polymerase is active, locus must be switched on.
Splicing site choice is governed by transcriptional environment of the gene.

Question 21

What is the process of splicing?

Answer 21

The 5’ site binds to the U1 snRNP, the Branch Point binds to the U2 snRNP.
These interact and associate with more snRNPs which forms a complex.
It allows a loop of intronic RNA to be formed which forms a phosphodiester bond across the two exons in the complex.
The snRNPs are released, mature mRNA is formed, and a loop of intron which is degraded in the cell.

Question 22

How can splicing be increased?

Answer 22

SRFs bind exonic splicing enhancers (ESE) and promote splicing by U2 auxiliary factor or the U1 snRNP.

Question 23

How can splicing be inhibited?

Answer 23

hnRNPs bind exonic splicing silencers (ESS) and inhibit splicing.

Question 24

What are the types of alternative splicing?

Answer 24

Alternative selection of: Promoters, Polyadenylation sites, Intron retaining mode, Exon cassette mode.
Additional exons = larger proteins = additional or altered function.

Question 25

What are the RNA polymerases?

Answer 25

Transcribe different non-overlapping subsets of genes.
Pol I - rRNA
Pol II - mRNA
Pol III - tRNA, rRNA, ncRNAs, miRNAs.

Question 26

What is the structure of the regulatory sequences?

Answer 26

5’ regulatory sequence controls transcription initiation.
The core promoter sequence, 100bp, determines the binding of RNA polymerase, and includes the Transcription Start Site.
Upstream there are more transcription factor sequences.

Question 27

What is RNA pol II?

Answer 27

A 12 subunit enzyme.
Requires an array of proteins and protein complexes to recognise the gene promoter and initiate transcription.
Transcription Factor IID can recognise the promoter.

Question 28

Why can RNA Pol II not recognise TSS?

Answer 28

Chromatin represses adventitious initiation - random expression of genes.
Can be methylated, so gene is not available.

Question 29

How does TFIID control transcription?

Answer 29

Core promoters contain sequences called a TATA box which is highly conserved, and TFIID can recognise and bind to and initiate transcription.

Question 30

What is TFIID?

Answer 30

A multi-protein complex, comprises of TATA binding protein (TBP), and TBP associated factors (TAFs).
TBP has specific binding site which forms hydrogen bonds with TATA box.
TFIID associates first then polymerase assembles around it.

Question 31

How does TBP distort the promoter DNA?

Answer 31

A saddle shaped protein which forms hydrogen bonds with the DNA.
This induces a 90 degree bend, which produces a signal and allows it to be recognised and for RNA polymerase to start.

Question 32

What is the holoenzyme?

Answer 32

Other TAF components bind - TFIIE and TFIIH.
TFIIH joining forms the holoenzyme and allows DNA to be activated by RNA polymerase.
TFIIE has helicase which opens the DNA up so transcription can take place.

Question 33

What is the initiation of transcription?

Answer 33

The Helicase unwinds the DNA template.
Protein Kinase phosphorylates the terminal DNA, which allows transcription, the spliceosome complex forms.
Pol II is released, TFIID remains to promote re-initiation so multiple proteins can be transcribed.

Question 34

What are enhancer sequences?

Answer 34

DNA sequence elements that are specifically associated with gene promoters.
Can be close or very far from the promoter, but folding of DNA can bring them into close association.
This increases rate of transcription.