Topic 5 Regulation of gene expression + W5 tutorial

Question 1

Bacteria can regulate metabolic pathways by:

Answer 1

1. Regulation of enzyme activity by feedback inhibition: controlled by allosteric regulation - Rapid response
2. Regulation of enzyme production by gene expression regulation: controlled by the operons - Longer term response

Question 2

Operon -

Answer 2

prokaryotic DNA segment that includes:
▪ The operator (segment of DNA which works as a regulatory (on-off) switch that controls a cluster of functionally related genes; consists of a specific sequence within a promoter of these genes)
▪ The promoter
▪ A group of functionally related genes

Question 3

Repressor -

Answer 3

a protein that switches off the operon: prevents gene transcription by binding to the operator and blocking RNA polymerase binding
- produced by a separate regulatory gene
- can be in an active or inactive form, depending on the
presence of other molecules

Question 4

Co-repressor -

Answer 4

A molecule that cooperates with a repressor protein to switch an operon off (operon inactivation)

Question 5

Negative gene regulation:

Answer 5

operons are switched off by the active form of the Repressor

Question 6

Repressible operons’ characteristics:

Answer 6

• Usually active
• Usually regulate gene expression of enzymes involved in anabolic pathways
• Their synthesis is repressed by high levels of the end product (corepressor) which activates the repressor

Example: the trp operon
E. coli can synthesize tryptophan, Trp operon contains genes of enzymes involved in tryptophan synthesis: Transcription is normally on but can be inhibited (repressed) when a small molecule (tryptophan) binds allosterically to a regulatory protein

ABSENCE of tryptophan = trp operon activated, repressor is inactive => can’t bind operator => genes for enzymes required for tryptophan synthesis are transcribed => tryptophan production

PRESENCE of tryptophan = trp operon is inactivated, trp - corepressor => binds to the trp repressor protein => repressor is activated => binds the operator => trp operon is inactivated => tryptophan production stops

!repressor is active only in the presence of its corepressor - tryptophan => trp operon is turned off (repressed) if tryptophan levels are high

Question 7

Inducible operons’ characteristics:

Answer 7

• Usually inactive
• Usually regulate gene expression of enzymes involved in catabolic pathways
• Their synthesis is induced by a chemical signal (inducer) which inactivates the repressor

Example: the lac operon
Lac operon: an inducible operon which contains genes that code for enzymes used in lactose metabolism (hydrolysis); Transcription is normally off but can be activated (induced) when a small molecule (lactose) binds allosterically to a regulatory protein

ABSENCE of lactose = lac operon isinactivated, lac operon is active by itself => binds to the operator => lac operon is inactivated => lactose hydrolysis stops

PRESENCE of allolactose - inducer => > inactivates repressor => The inducer turns the lac operon on => genes for enzymes involved in lactose hydrolysis are transcribed => Lactose hydrolysis starts => lactose is broken down to glucose and galactose

Question 8

Inducer -

Answer 8

molecule that binds and inactivates the repressor

Question 9

Positive gene regulation

Answer 9

operons are switched on by the active form of the activator - a stimulatory protein, for ex: Catabolite Activator Protein (CAP) in E. coli which enhances transcription of the lac operon

Question 10

positive gene regulation: lac operon in bacteria affected by glu lvls

Answer 10

* Low glucose levels: Increase in levels of cAMP => CAP is activated by binding to cAMP => Activated CAP attaches to the promoter of the lac operon => increases the affinity of RNA polymerase => accelerates
transcription of the lac operon (genes producing proteins involved in hydrolysis of lactose to glucose and galactose)

* High glucose levels
Decrease in levels of cAMP => CAP detaches from the lac operon => Decreased affinity of RNA polymerase and decreased (low) transcription of the lac operon (genes producing proteins involved in hydrolysis of lactose to glucose and galactose)

Question 11

Role of Gene expression in eukaryotes: (2)

Answer 11

1. Regulates development
2. Is responsible for cell specialization (differentiation): expression of different genes by cells with the same genome => different cell types produced

Question 12

How can eukaryotic gene expression can be regulated at any stage?

Answer 12

1. Regulation of chromatin structure
   - Histone acetylation
   - DNA methylation
2. Regulation of transcription initiation
3. Post-transcriptional regulation
   - RNA processing
   - mRNA degradation
   - initiation of translation
   - protein processing and degradation

Question 13

Histone acetylation: exact process

Answer 13

The N- terminus of each histone molecule in a nucleosome protrudes outward from the nucleosome
- Acetyl groups (-COCH3 ) are attached to (+) charged lysines in histone tails => lysines are acetylated, their positive charges are neutralized and the histone tails do not bind to neighboring nucleosomes => Chromatin has a looser structure => Activation of transcription
- implemented by Histone acetylation enzymes (Histone acetylation transferaze)

Question 14

Histone deacetylation: exact process

Answer 14

Removal of Acetyl groups (-COCH3) restores the histone (+) charge => increased binding to neighbouring nucleosomes => inactive form of chromatin => inactivation of transcription

implemented by histone deacetylases (HDACs)

Question 15

Histone methylation:

Answer 15

Addition of methyl groups (-CH3) (nonpolar group/ neither negative nor positive) in an amino acid (lysine or arginine) in the histone => Chromatin condensation => Gene expression inactivation

!*Even though histone methylation is in general associated with transcriptional repression, methylation of some lysine and arginine residues of histones results in transcriptional activation.

Question 16

Phosphorylation

Answer 16

Addition of a phosphate group to an amino acid which is next to a methylated amino acid => decondensation of chromatin => activation of transcription

Question 17

DNA methylation:

Answer 17

The addition of methyl groups (-CH3) to certain bases in DNA (usually cytosine) => reduced transcription
It can cause long-term inactivation of genes involved in
cellular differentiation. Comparison of the same genes in different tissues shows that the genes are usually more heavily methylated in cells in which they are not expressed

Question 18

Organization of a Typical Eukaryotic Gene (3)

Answer 18

1. Promoter:
   - a DNA sequence where RNA polymerase II and transcription factors bind
   - Present in each eukaryotic gene
   - Located upstream of the gene
   - Includes TATA box
2. Control elements:
   - Segments of non-coding DNA that regulate transcription by binding to transcription factors
   o Proximal control elements: located close to the promoter
   o Distal control elements (grouped together as enhancers): located far away from a gene or even within an intron
3. Transcription factors:
   - Proteins which help RNA polymerase II to initiate transcription
   - Interact with specific control elements => regulate
   transcription of particular genes

Control elements + transcription factors = regulation of gene expression in diff cell types

Question 19

2 types and Roles of Transcription Factors

Answer 19

Transcription factors help RNA polymerase II to initiate transcription and are essential for the transcription of all protein-coding genes

* 2 types of specific transcription factors:
▪ Activators: transcription factors that bind to an enhancer and stimulate specific gene transcription
▪ Repressors: transcription factors that inhibit transcription and expression of a particular gene

Question 20

Enhancers and Specific Transcription Factors (TFs)

Answer 20

Activators bind to an enhancer (control element) to stimulate specific gene transcription => Bound activators cause recruitment of mediator proteins => Recruitment of general TFs which bind to TATA box within the promoter => Recruitment of RNA polymerase II which binds to the promoter => Activation of gene transcription

Question 21

Activators have 2 domains:

Answer 21

DNA-binding domain and an activation domain (activates transcription)

Question 22

Types of Transcription Factors (2):

Answer 22

Specific transcription factors (Activators):
- Unique for each gene (only common for the functionally related genes that need to be coexpressed)
- Once these TFs bind to the control elements (e.g. enhancers), they cause recruitment and binding of the general transcription factors to the TATA box

General transcription factors:
- Common for all the genes
- Bind to the TATA box in order to induce RNA polymerase II binding to the promoter

Question 23

Co-expression of genes in prokaryotes vs eukaryotes:

Answer 23

Prokaryotes:
* Functionally related genes of a prokaryotic operon are regulated by the same promoter
* Production of polycistronic mRNA molecule (encodes more than one polypeptide) => co-expressed

Eukaryotes:
* Each eukaryotic gene has its own promoter and control elements
* Production of monocistronic mRNA (encodes for only one polypeptide)
* Functionally related genes have the same control elements and activators even if located on different chromosomes
* Activators recognise specific control elements and promote simultaneous transcription of genes => co-expressed

Question 24

Post-transcriptional regulation -

Answer 24

regulatory mechanisms that operate at various stages after transcription => provide rapid regulation of gene expression in response to environmental changes

Question 25

Clinical Example of Alternative RNA splicing:

Answer 25

***β-thalassaemia***: some types due to abnormal splicing of the β-globin gene

Question 26

mRNA degradation as post-transcriptional regulation:

Answer 26

The **life span of mRNA** molecules in the cytoplasm is a **key to determining protein synthesis** * Eukaryotic mRNA is more long lived than prokaryotic mRNA * The mRNA life span is determined in part by sequences in the *3’-untranslated region (3'-UTR)* * Nucleases and non-coding RNAs induce mRNA degradation => Inhibition of gene expression

Question 27

what induces mRNA degradation to inhibit gene expression?

Answer 27

Nucleases and non-coding RNAs

Question 28

Non-coding RNAs can:

Answer 28

- inhibit initiation of translation - induce mRNA degradation

Question 29

What post-translational modifications of polypeptide happen in RER? (3/4)

Answer 29

1. **Polypeptide cleavage**: some polypeptides are activated by enzymes that cleave them (e.g. insulin) 2. **Protein folding (tertiary structure)**: e.g. disulphide bond formation 3. **Subunit assembly (quaternary structure)**: some polypeptides come together to form the subunits of a functional protein (e.g. hemoglobin) 4. **Chemical modifications**: addition of chemical groups to proteins => formation of glycoproteins, lipoproteins, ONLY FOR SOME POLYPEPTIDES

Question 30

What post-translational modifications of polypeptide happen in Golgi? (1)

Answer 30

**Chemical modifications**: addition of chemical groups to proteins => formation of glycoproteins, lipoproteins, mostly in Golgi, some in RER

Question 31

Protein degradation: Proteasome

Answer 31

***Proteasomes*** - giant protein complexes that bind to protein molecules and degrade them (size= 26S) to mark a particular protein for destruction, the cell attaches the small protein ***ubiquitin*** to the protein e.g.* cyclins in the cell cycle*, *non-functional (misfolded) proteins* are also attached to ubiquitin (ubiquitination) => Ubiquitinated proteins are targeted to the proteasome for degradation

Question 32

where are Long lived proteins degraded?

Answer 32

in the lysosome

Question 33

Coding DNA percent?

Answer 33

Only 1.5% of the DNA codes for proteins (Coding DNA)

Question 34

Function of noncoding RNAs:

Answer 34

regulate mRNA translation and chromatin configuration => regulate gene expression

Question 35

MicroRNAs (***miRNAs***):

Answer 35

Small **single-stranded RNA** molecules (20-25bp) that can bind to mRNA and **can degrade mRNA but mostly block its translation** fn: Mostly inhibits translation miRNA - incomplete base pairing with many different mRNAs => can target various mRNAs and inhibit expression of various genes

Question 36

Small interfering RNAs (***siRNAs***):

Answer 36

Small **double-stranded RNA molecules** (20-25 bp) that bind to the mRNA and **cause RNA interference (RNAi) - inhibition of gene expression by RNA molecules** fn: Mostly causes mRNA degradation siRNA - complete base pairing with a specific mRNA => can inhibit expression of specific genes only (highly specific)

Question 37

siRNAs' fns: (2)

Answer 37

1. **Chromatin modification:** induce heterochromatin formation => can block large regions of the chromosome by inhibition of transcription and gene expression 2. **Transcription regulation:** inhibit transcription of specific gene

Question 38

Epstein-Barr virus | type and association

Answer 38

DNA associated w/ Burkitt’s lymphoma, Nasopharyngeal carcinoma

Question 39

Human Papilloma viruses | type and association

Answer 39

DNA associated w/ Cervical carcinoma, warts

Question 40

Hepatitis B virus | type & association

Answer 40

DNA associated w/ Hepatocellular carcinoma

Question 41

HTLV-I | type & association

Answer 41

RNA retrovirus associated w/ Adult T-cell leukaemia lymphoma

Question 42

Acutely transforming oncogenic retroviruses:

Answer 42

-e.g. *Avian Erythroblastosis Virus (v-erb-b2), Avian Myelocytomatosis Virus (v-myc)* - cause tumours by transduction of the viraloncogene (***v-Onc***): virus genome contains an oncogene => insertion into the genome of the cell => uncontrolled proliferation of cells => carcinogenesis **viral oncogene is constitutively active**

Question 43

Non-defective(slowly transforming) oncogenic retroviruses:

Answer 43

e.g. *Mouse Mammary Tumour Virus (MMTV), Avian Leukosis Virus (ALV)* - cause tumours by **insertional mutagenesis**: virus genome does not containan oncogene => inserts itself upstream of ***c-myc*** (cellular proto-oncogene responsible for normal cell proliferation) => strong viral promoter causes uncontrolled cell proliferation => carcinogenesis

Question 44

Oncogenes -

Answer 44

Genes found *in viral or cellular genomes* that t**rigger the molecular events that can lead to cancer** => oncogenes induce uncontrolled cell division and => cancer development

Question 45

Tumour suppressor genes -

Answer 45

Genes whose **protein product inhibits cell division** => tumour suppressor genes prevent uncontrolled cell division and therefore prevent cancer development **Inactivation of tumour suppressor genes leads to cancer**

Question 46

Oncogenes and proto-oncogenes

Answer 46

***Oncogenes*** - cancer-causing genes ***Proto-oncogenes*** - the corresponding normal cellular genes that are responsible for normal cell growth and division (*e.g. growth factors, receptors, intracellular molecules of signalling pathways*) **Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle**

Question 47

Proto-oncogenes can be converted to oncogenes by:

Answer 47

1. Movement of DNA within the genome 2. Amplification of a proto-oncogene 3. Mutations in the proto-oncogene or its control elements

Question 48

**Movement of DNA within the genome**

Answer 48

by translocation / transduction, e.g. in *Chronic Myelogenous Luekemia (CML):* fusion between gene **ABL (chromosome 9)** and gene **BCR (chromosome 22)** => an oncogene = a protein with enhanced tyrosine kinase activity that leads to increased cell division and therefore tumorigenesis. 95% of cases of Chronic Myeloid Leukaemia (CML) have detectable Ph chromosome. DNA may be inserted downstream of an active promoter => Transcription of the genes that it encodes may increase; Inserted DNA may contain an active promoter (e.g. viral promoter) => Transcriptional activation of genes

Question 49

Amplification of a proto-oncogene

Answer 49

increased number of copies of the gene, *e.g. EGFR-2 amplification in HER-2 positive breast cancers* => Increased activation of the MAPK signaling pathway => Increased cellular proliferation

Question 50

Trastuzumab (Herceptin) -

Answer 50

monoclonal antibody that targets HER2 (targeted therapy)

Question 51

Mutations in the proto-oncogene or its control elements

Answer 51

gain-of-function mutations: ▪ Increased gene expression of the proto-oncogene ▪ Constitutively active oncogene (*e.g. Ras*) => production of the Ras oncogene => hyperactive Ras protein => increased cell division => cancer

Question 52

Mechanism of inactivation of tumour suppressor genes:

Answer 52

Loss-of function mutations: inactivating mutations within tumour-suppressor genes → inactive tumor suppressor genes→ inactive proteins → cancer development or Insertional mutagenesis: insertion of viral genome into host cell DNA → inactive tumor suppressor genes→ inactive proteins → cancer development

Question 53

Roles of Tumor-suppressor proteins (3):

Answer 53

▪ Inhibit cell-signaling pathways → > inhibit the cell cycle or induce apoptosis (***e.g. Rb and p53 proteins***) ▪ Repair damaged DNA (***e.g. BRCA-1***) ▪ Control cell adhesion

Question 54

p53 - ‘the gatekeeper of the genome’, why?

Answer 54

p53 **prevents a cell that has DNA damage from passing on mutations to its daughter cells by replicating**, activated by DNA damage => causes cell cycle arrest *at G1 phase* Mutations in the p53 gene prevent cell cycle arrest => Uncontrolled cell proliferation of damaged cells => Cancer

Question 55

colorectal cancer - which inherited mutations of tumour-supressor genes?

Answer 55

Inherited mutations in the tumour-suppressor gene ***adenomatous polyposis coli (APC)*** are common in individuals with colorectal cancer

Question 56

breast cancers, also ovarian cancers & pancreatic cancer in males - which inherited mutations?

Answer 56

Mutations in the ***BRCA1 or BRCA2*** genes are found in at least half of inherited breast cancers

Question 57

HER-2 -

Answer 57

oncogene, growth factor receptor

Question 58

Ras -

Answer 58

oncogene, MAPK pathway protein

Question 59

p53 -

Answer 59

Tumour suppressor gene, DNA repair, G1 arrest

Question 60

Rb -

Answer 60

Tumour suppressor gene, G1 arrest

Question 61

BRCA-1 and BRCA-2 -

Answer 61

Tumour suppressor genes, ds DNA repair