Control of Eukaryotic Gene expression

Question 1

What is the common and most important control point for gene expression in all organisms

Answer 1

transcription

Question 2

A few facts about transcription control

Answer 2

1. It is often in response to signals coming from outside the cell, such as hormones or other signalling molecules.
2. It includes a few regulatory mechanisms, namely regulation of gene accessibility (histone acetylation and DNA methylation) and initiation of transcription

Question 3

How can a chromatin be organised

Answer 3

Chromatin has the appearance of beads on a string. Each bead is a nucleosome comprising DNA complexed with a histone octamer (RECAP

Chromatin can be organised into

1. Euchromatin which is diffused and available for transcription
2. Heterochromatin which is highly condensed and transciptionally inactive

Question 4

What does condensation do?

Answer 4

It prevents transcription factors and RNA polymerase from gaining access to the promoter of a specific gene, thus inactivating transcription of that gene

Question 5

What are histone tails

Answer 5

The N-terminus of each histone in nucleosome protrudes outwards from the nucleosome. Thus, for every nucleosome, 8 histone N-temini protrude outwards and these protrusions are known as histone tails

Question 6

What are some features of histone tails

Answer 6

1. rich in lysine residues, which are positively-charged. This allows them to strongly interact with the negatively-charged phosphate groups of the DNA backbone and increase the affinity of DNA for the nucleosome surface
2. The tails are accessible to various modifying enzymes, which catalyse the addition or removal of specific chemical groups.

These chemical groups in turn alter the tightness of DNA winding around the histones, thus altering the ease of transcription initiation

Question 7

Describe the acetylation of histone tails

Answer 7

1. Positively charged lysine residue in the histone tails can be acetylated by histone acetyltransferase (HATs)
2. When lysines are acetylated, their positive charges are neutralised. The affinity of the histone complex for DNA is reduced
3. Consequently, the chromatin becomes more diffused/ less compact. Control regions of genes would be exposed to transcription factors and RNA polymerase

Question 8

describe the deacetylation

Answer 8

1. Histone deacetylases (HDACs) catalyse the deacetylation of acetylated lysine residues in histone tails
2. Lysine residues regains their positive charges, resulting in an increase in the affinity of the histone complex of DNA
3. Consequently, the chromatin becomes more compact and prevents access of transcription factors and RNA polymerase to the control region of genes.

Question 9

Give an overview of DNA methylation

Answer 9

DNA can be covalently modified by the addition of methyl groups to specific nucleotides after DNA replication. DNA methylation in vertebrate DNA is restricted to cytosine nucleotides in the sequence 5’-CG-3’ (CpG dinucleotides)

This process is catalysed by DNA methyltransferase

CpG dinucleotides are clustered to form CpG islands, which are usually found in the promoter regions of many genes. Methylation of cytosines within a gene’s promoter sequence prevents transcription of the gene.

Question 10

What are the 2 mechanisms that explain how DNA methylation represses gene expression

Answer 10

1. Methylation changes the 3D conformation of DNA and thus, prevents the binding of transcription factors to the promoter. Transcription initation is prevented
2. Methylated DNA serves as recognition signals for methyl-CpG-binding proteins (MeCP) that in turn recruit other proteins such as histone deacetylases (HDACs).
   
   • The HDACs modify chromatin in the region of the CpG island such that it becomes more condensed.
   • This prevents binding of transcription factors and RNA polymerase. Transcription initation is prevented

Question 11

What does control of transcription initiation determine

Answer 11

1. Whether or not genes are expressed
2. Quantity of encoded mRNAs
3. Consequently, the quantity of proteins produced

Question 12

What is a TIC

Answer 12

For transcription of a gene to begin, general transcription factors and RNA polymerase must assemble at the promoter to form a transcription initiation complex (TIC)

Question 13

How do you achieve max transcription rate of a gene

Answer 13

The interaction of specific transcription factors and distal control elements (enhancers or silencers) with the general transcription factors and RNA polymerase to form a stable TIC.

Question 14

What are some important intergenic DNA sequences

Answer 14

1. origin of replication
2. centromeres
3. telomeres

Question 15

What are distal control elements

Answer 15

It consists of enhancers and silencers that can exert their effects when located hundreds or even thousands of bp upstream or downstream of the transcription start site. Some may also be locarted within an intron

Enhancers are DNA sequence that bind specific regulatory proteins known as activators to increase transcription rate

Silencers interact with other specific regulatory proteins proteins known as repressors to decrease transcription rate.

Both can function in an orientation-independent fashion

Question 16

Fun fact on page 11

Hint control elements binding and specificity

Answer 16

The control elements bind different transcription factors which are proteins

The particular combinations of control elements and transcription factors are specific to each gene and result in different transcription rate in different cell types (spatial specificity) or at different stages of development (temporal specificity)

Question 17

What is a transcription factor

Answer 17

A regulatory protein that binds to DNA and affects transcription of genes

Question 18

What are the 2 groups of specifc transcription factos

Answer 18

• Activators (proteins) bind to enhancers (DNA sequence), triggering a series of interactions that results in an increased rate of transcription
• Represssors (proteins) bind to silencers (DNA sequence), triggering a series of interactions that results in a decreased rate of transcription

Question 19

What are the 2 binding domains in specific transcription factors

Answer 19

1. A DNA binding domain - part of the protein’s 3D structure that binds DNA
2. One or more protein binding (activation) domains, which binds other regulatory proteins or components of the transcription machinery, facilitating the protein-protein interactions that result in gene transcription

Question 20

Name the first mechanism that repressers can act to inhibit transcription

Answer 20

Competitive DNA binding

• Activator proteins and repressor proteins compete for binding to the same regulatory DNA sequence

Question 21

Name the second mechanism that repressers can act to inhibit transcription

Answer 21

Masking the activation surface

• Both proteins bind DNA, but the repressor prevents the activator from interacting with the general transcription factors

Question 22

Name the third mechanism that repressers can act to inhibit transcription

Answer 22

The repressor blocks assembly of the general transcription factors

Question 23

Name the fourth mechanism that repressers can act to inhibit transcription

Answer 23

The repressor recruits a chromatin remodelling complex

• The chromatin remodelling complex returns the nucleosomal state of the promoter region to its pre-transcriptional form

Question 24

Name the fifth mechanism that repressers can act to inhibit transcription

Answer 24

The repressor attracts a histone deacetylase to the promoter

• The repressor reverses histone transcription initiation

Question 25

Name the sixth mechanism that repressers can act to inhibit transcription

Answer 25

The repressor attracts a histone methyl transferase

• Histone methyltransferase modifies certain positions on histones by attaching methyl groups;
• The methylated histones, in turn, are bound by proteins that maintain the chromatin in a transcriptionally silent form

Question 26

Why is post-transcriptional modification important

Answer 26

All pre-mrna must go through post-transcriptional modification to produce functional mature RNA molecules for export to the cytosol

Question 27

What does post-transcriptional modification involve`

Answer 27

1. Modification of the 5’ end of the nascent RNA chain by capping with 7-methylguanosine triphosphate to form a 5’ cap
2. RNA splicing occurs after the release of pre-mRNA from the RNA polymerase

• Defined as the removal of introns while remaining exons are ligated together
• requires the hydrolysis of ATP and is carried out at splice sites by spliceosomes

3. Modification of the 3’ end by polyadenylation.
   * which is where a series of adenine nucleotidss are added to the 3’ end by an enzyme called poly(A) polymerase. The resulting 3’poly(A) tail is about 200 nucleotides long

Question 28

What are spliceosomes

Answer 28

A large complex comprising of several subunits known as small nuclear ribonucleoproteins (snRNPs)

Question 29

Describe the process of mRNA splicing

Answer 29

1. Cleavage of the 5’ splice site and joining of intron to a branch point within the intron. This reaction yields a lariat-like intermediate, in which the introns forms a loop
2. Cleavege at the 3’ splice site and simultaneous ligation of the exons, resulting in an excision of the intron as a lariat-like structure
3. DNA sequences at the 5’ and 3’ ends of an intron serve as recognition siters for the spliceosome to bind

Question 30

What does a mature mRNA consists of

Answer 30

1. 5’ cap
2. 5’ UTR
3. Protein coding region
   
   • Consists of a series of codons representing the amino acid sequence of the protein, starting with start codon (AUG) and ending with a stop codon (UAA, UAG, UGA)
4. 3’ UTR
5. 3’ poly A tail

Question 31

How can a single gene synthesis different polypeptides

Answer 31

pre-mRNA transcribed from one gene can be processed in more than one way, leading to formation of different mature mRNAs, each containing a different combination of exons

This use of different splice sites to allow exons to join together in different combinations is known as alternative splicing

Question 32

What does the stability of mRNA determine

Answer 32

It determines the duration for which translation can occur. The less stable (i.e. the more rapidly an mRNA molecule is degraded), the less time is available for it to be translated

In general, the mRNAs in eukaryotic cells are more stable than those in prokaryotic cells, and thus have a longer half life

Question 33

What is stability of an mRNA affected by

Answer 33

1. Length of poly(A) tail
   
   • mRNAs with longer poly (A) tails tend to be more stable
2. Stabilising/destabilising sequence in the 3’ UTR
   
   • these sequences contain binding sites for specifc proteins that increase or decrease the rate of poly (A) tail shortening

Question 34

What are the two mechanisms that exist for the eventual decay of eukaryotic mRNA

Answer 34

Both begin with the gradual shortening of the poly-A tail by an exonuclease, a process that starts as soon as the mRNA reaches the cytosol.

In a broad sense, this poly-A shortening acts as a timer that counts down the lifetime of each mRNA.

Once the poly-A tail is reduced to critical length, the 2 pathways diverge

• The 5’ cap is removed (decapping) and the ‘exposed’ RNA is rapidly degraded from its 5’ end

or

• The mRNA continues to be degraded from the 3’ end, through the poly-A tail, into the coding sequences

Both processes can occur together on the same molecule. Poly-A shortening controls the half-life of most eukaryotic mRNAs

Question 35

What is another specialize mechanism that degrades some mRNA

Answer 35

An endonuclease (specific nucleases that cleaves the mRNA internally) effectively decaps one end and removes the poly-A tail from the other so that both halves are rapidly degraded.

mRNA that are destroyed in this way carry specific nucleotide sequences, often in the 3’ UTR that serves as recognition sequences for these endonucleases. This strategy makes it simple to tightly regulate the stability of these mRNAs by blocking or exposing the endonuclease site in response to extracellular signals

Question 36

What is cytoplasmic poly-A tail addition

Answer 36

Modifications made to specific mRNAs present in the cytoplasm to promote translation. Achieved by lengthening their poly (A) tails by cytoplasmic poly (A) polymerases.

Question 37

What are eIFs

Answer 37

The initiation of translation in both eukaryotes and prokaryotes is dependent on translation factors in addition to the small and large subunits of ribosome, these translations factors are eukaryotic initiation factors (eIFs)

eIFs are involved in:

• scanning the mRNA for the start codon AUG,
• locating the binding site of initiator tRNA to the AUG codon and
• forming the translation initiation complex at the 5’ mRNA region

By varying the abundance and activity of these factos it is possible to affect the rate of translational initiation. However this has a global effect on overall translational activity, rather than on the rate of translation of specific mRNAs

Question 38

What are translational repressors

Answer 38

Molecules that bind to various regions of mRNA molecule, usually the 5’ and 3’ UTRs, and interfere with the initiation of translation by blocking the attachment of ribosomes or other translational initiation factors

This molecules decreases the rate of translation initiation as a result

Question 39

What are the other sites where translation may be initiated instead of the first AUG codon

Answer 39

1. Second or subsequent AUG

• Small ribosomal subunit could skip the first AUG codon and use the second or subsequent AUG to initiate translation
• This is known as “leaky scanning” and results in proteins that vary in their N-terminal sequence

2. Initiation of translation in the middle of mRNA

• Eukaryotic translation usually begeins at the 5’ end of the mRNA molecule, since 5’ cap recognition is required for assembly of the initiation complex
• The internal ribosome entry site (IRES) is a specialised nucleotide sequence that allows for translation initiation in the middle of an mRNA sequence in a cap-independent manner, since the need for a 5’ cap is bypassed.
• A protein with a different structure is produced with this mechanism

Question 40

What is RNA interference

Answer 40

A group of short non-coding RNAs (3 types, one of which is micro RNAs or miRNAs) carry out RNA interference.

These RNA locate their target through RNA-RNA base-pairing, and they generally cause reductions in gene expression

Question 41

Summarise the action of miRNA

Answer 41

1. RNA transcripts (synthesised by transcribing miRNA-coding genes) fold back on themselves, forming a hairpin structure held together by hydrogen bonds
2. They are processed by an enzyme called Dicer, which cuts the double-stranded RNA into smaller fragments.
3. One strand of the double-stranded RNA fragment is degraded by protein complex known as RNA-inducing silencing complex (RISC). The remaining strand bind to RISC to form miRNA-protein complex
4. This miRNA strand then binds to mRNA molecules that have the complementary sequence
5. The miRNA-protein complex then inhibits translation by blocking formation of the translation initiation complex or degredation of the mRNA

Question 42

Post-translational modification significance

Answer 42

Many proteins have limited lifespan. Some proteins that trigger metabolic changes in cells are broken down by proteasomes within a few minutes or hours

This regulation allows a cell to

• Adjust the kinds and amounts of its proteins in response to changes in its environment
• maintain its proteins in working order