chapter 7: control of eukaryotic gene expression

Question 1

what are housekeeping genes?

Answer 1

• genes that must be expressed in all cells at all times
• these genes code for proteins responsible for routine metabolic functions

Question 2

what are the successive levels in which eukaryote DNA is packed into chromosomes?

Answer 2

• the packing of DNA into chromosomes occurs via coiling and folding in successive levels

1. linear DNA double helix
2. 10nm nucleosome
3. 30nm chromatin fibre/ solenoid
4. 300 nm looped domains

chromosome!

Question 3

how eukaryote DNA is packed into chromosomes: NUCLEOSOME

Answer 3

nucleosome is the most basic level of packing of DNA into chromosomes occurs eukaryotes

• a nucleosome consists of negatively charged DNA wound around positively charged histone protein core
• each core is also known as a histone octamer as it contains 8 histone proteins
• histone proteins have high proportion of positive charged amino acids (lysine and arginine), and they bind, and they bind tightly to negatively charged DNA
• the histone tails extend outwards from the nucleosome
• adjacent nucleosomes are connected by linker DNA and H1 histones, to give the appearance of ‘ beads-on-a-string’

Question 4

how eukaryote DNA is packed into chromosomes: 30NM CHROMATIN FIBRE/ SOLENOID

Answer 4

• interactions between histone tails, linker DNA and H1 histone
• cause the string of nucleosomes to coil to form a chromatin fibre that is 30nm wide in diameter
• which is a solenoid

Question 5

how eukaryote DNA is packed in chromosomes: LOOPED DOMAINS

Answer 5

• the 30nm chromatin fibre folds to form looped domains
• that are attached to a base of scaffolding (non-histone) proteins
• non histone proteins known as scaffold proteins are used to condense the solenoid to form looped domains

Question 6

how is eukaryote DNA packed into chromosomes occurs eukaryotes: CHROMOSOMES

Answer 6

• only during nuclear and cell division, the looped domains coil and fold further
• compacting all the chromatin to highly condensed metaphase chromosomes
• that can be seen under the light microscope

Question 7

how is the packing in euchromatin different from the packing in heterochromatin?

Answer 7

euchromatin:
- loosely coiled/ less condensed/ less compact

heterochromatin:
- tightly coiled/ heavily condensed/ compact

Question 8

compare how transcriptionally active euchromatin and heterochromatin are

Answer 8

euchromatin: (loosely coiled)
- transcriptionally active because RNA polymerase and general transcription factors are able to bind to promoter of genes

heterochromatin: (tightly coiled)
- transcriptionally inactive because RNA polymerase and general transcription factors are unable to bind to the promoter of genes
- packing of DNA into heterochromatin, where DNA is highly condensed is typically for the long term inactivation of genes
- where gene expression is repressed for a long period of time/ permanently

Question 9

what are euchromatin and heterochromatin composed of?

Answer 9

euchromatin:
- 30nm fibres and looped domains

heterochromatin:
- 30nm fibres and looped domains and additional proteins that help in compaction

Question 10

what happens during histone acetylation?

Answer 10

• acetylation is the addition of acetyl groups to lysine in the histone tails
• reaction is catalysed by histone acetyl transferase
• when the lysine residues on histone tails are acetylated
• their positive charges are neutralised
• this causes fewer interaction of histone tails with the negatively charged phosphate groups of DNA that is being wrapped around the histones
• and fewer interactions of histone tails with neighbouring nucleosomes
• chromatin structure becomes less compact and condensed
• RNA polymerase and transcription factors can bind to the promoters of genes in the acetylated region to form transcription initiation complex
• allowing transcription to occur/ gene to be transcriptionally active

Question 11

what does the deacetylation of histone tails cause?

Answer 11

deacetylation of histone tails by histone deacetylase causes:
- chromatin structure to become more compact and condensed
- RNA polymerase and transcription factors cannot bind to the promoters of genes in the deacetylated region
- transcription initiation complex cannot form
- transcription is prevented/ gene is transcriptionally inactive

Question 12

what happens during histone methylation?

Answer 12

• methylation is the addition of methyl groups to lysine and arginine amino acid R groups in histone tails
• this reaction is catalysed by histone methyltransferase

methylated histones can:
- attract proteins to bind to the histones
- causing DNA to wound tightly around the histones resulting in condensation of the chromatin
- RNA polymerase and transcription factors cannot bind to the promoters of of genes in the methylated region
- transcription initiation complex cannot form
- transcription is prevented/ gene is transcriptionally inactive

Question 13

histone acetylation > ________
histone deacetylation > ________
histone methylation > ________
histone demethylation > ________
DNA methylation > ________

Answer 13

histone acetylation > allows transcription to occur
histone deacetylation > prevents transcription
histone methylation > prevents transcription
histone demethylation >allows transcription to occur
DNA methylation > prevents transcription

Question 14

what happens when DNA methylation occurs?

Answer 14

• DNA methylation is the addition of methyl groups to DNA
• the reaction is catalysed by DNA methyltransferase

methylated DNA:
- attracts other proteins
- which in turn recruit histone deacetylases enzymes that remove acetyl groups from histone tails
- this makes the chromatin more compact and condensed
- RNA polymerase and transcription factors cannot bind to the promoters of genes in the methylated region
- transcription initiation complex cannot form
- transcription is prevented and gene is transcriptionally inactive

Question 15

what are control elements?

Answer 15

• they are regulatory DNA sequences that are non-coding
• they serve as binding sites for RNA polymerase and transcription factors which are proteins that regulate transcription and the level of gene expression

Question 16

CONTROL ELEMENTS
what is a promoter?

Answer 16

• the site where transcription of a gene is initiated
• a DNA sequence where RNA polymerase and general transcription factors bind to and initiate transcription
• for eukaryotic genes, the promoter commonly contains the TATA box, with DNA sequence of 5’-TATAAA-3’
• in eukaryotes, each gene is under the control of a single promoter

Question 17

CONTROL ELEMENTS:
what are enhancers/ silencers?

Answer 17

• a DNA sequence where specific transcription factors bind to control the rate of transcription
• enhancer: a control element that increases transcription rate
• silencer: a control element that decreases / inhibit rate of transcription
• these DNA sequences may lie far away from the promoter
• however, they can still control the rate of transcription because the ** DNA can bend** to bring the enhancer/ silencer close to the promoters of genes
• these sequences are very important for tissue specific gene expression

Question 18

what are introns and exons?

Answer 18

• sequences of DNA nucleotides in eukaryotic genes is usually not continuous, it is split into exons and introns
• coding DNA segments in a gene are called extrons because they are eventually EXpressed
• non-coding DNA segments in a gene are called introns because they are found between exons, hence INtervening the coding regions

Question 19

what is a poly-A-signal sequence?

Answer 19

• it is a non coding sequence
• a DNA sequence that is transcribed into an RNA sequence on the mRNA
• this signals addition of poly-A-tail to the 3’ end of mRNA transcript during RNA processing

Question 20

how does masking the activation domain on the activator inhibit transcription?

Answer 20

• both repressor and activator bind to DNA
• but the repressor also binds to the activation domain on the activator
• this prevents activator from binding to and stabilising the transcriptional initiation complex, preventing the transcription rate from being increased

Question 21

what are transcription factors? and what are the two types of transcription factors?

Answer 21

transcriptional regulation in eukaryotes involves proteins called transcription factors that bind to:
- control elements
- and or other transcription factors/ RNA polymerase

• to facilitate or inhibit the formation of transcription initiation complex on the promoter
• the two types: general transcription factors and specific transcription factors

Question 22

what is the structure of a transcription factor?

Answer 22

transcription factors have two or more binding domains

1. DNA binding domain is the region on the TF that binds to specific DNA sequences
   eg. promoter, enhancer, silencer
   the DNA binding domain is complementary in shape to the specific DNA sequence because:
   - different sequence of bases on DNA have different shapes
   - the major and minor grooves on DNA also have an effect on shape of that specific DNA
2. protein binding/ activation domain is the region on the TF that binds to other transcription factors or proteins
   - for binding **between different TFs **to occur, the protein binding domain of one TF must be complementary in shape to the protein binding domain of another TF/ RNA polymerase

Question 23

describe the mechanism of the initiation of transcription at the promoter

Answer 23

1. a general transcription factor binds to the TATA box at the promoter
2. binding of the general transcription factor recruits other **additional general transcription factors **and RNA polymerase to assemble at the promoter
   - forming the transcription initiation complex (which is relatively unstable at this point)
3. this results in low rate of transcription
   - (Without additional regulatory factors (like enhancers or activators), transcription occurs at a basal or low level.)

Question 24

what does the formation of a transcription initiation complex ensure?

Answer 24

• RNA polymerase is positioned correctly at promoter
• DNA double helix is unwound and the DNA molecule is unzipped to expose the template DNA strand

Question 25

how do activators enhance the rate of transcription?

Answer 25

activators once bound to enhancers work by: - accelerating and stabilising formation of transcription initiation complex - modifying chromatin structure

Question 26

what is the mechanism of activators enhancing the rate of transcription?

Answer 26

1. activators bind to enhancers 2. a DNA bending protein binds to DNA, causing the DNA to bend to bring the bound activators close to the promoter - where RNA polymerase, general transcription factors and other proteins like mediator proteins are recruited to 3. the activators bind to one or more basal transcriptionally factors and or mediator proteins and RNA polymerase, helping to accelerate and stabilise the formation of the transcription initiation complex on the promoter - highly increased rate of transcription of the gene - activators may recruit proteins and complexes such as histone acetyltransferase that loosens chromatin structure> which promotes the formation of TIC

Question 27

what happens when activators modifies chromatin structure?

Answer 27

some activators act indirectly by modifying chromatin structure once bound, activators may recruit - histone modification enzymes (eg. histone acetyl transferase) - chromatin remodeling complexes - histone chaperone proteins - this results in the chromatin structure to be loosened, allow RNA polymerase and general transcription factors to bind at the promoter - promoting the formation of the transcription initiation complex

Question 28

what are the four ways that can inhibit gene expression?

Answer 28

1. competitive DNA binding 2. masking of the activation domain on activator 3. block assembly of transcription initiation complex 4. modifying chromatin structure

Question 29

how does competitive DNA binding inhibit transcription?

Answer 29

- enhancer and silencer DNA sequence overlap - activator and repressor proteins compete to bind to the same sequence of DNA - activators may be prevented from binding to enhancer sequence, preventing the transcription rate from being increased

Question 30

how does blocking the assembly of transcription initiation complex inhibit transcription?

Answer 30

- the repressor binds with some general transcription factors - this blocks the assembly of the transcription initiation complex on the promoter

Question 31

how does modifying the chromatin structure inhibit transcription?

Answer 31

- once bound, repressions may recruit - chromatin remodeling complexes - histone deacetylases - histone methyltransferase - this results in the chromatin structure to e more compact - preventing RNA polymerase and general transcription factors from binding at the promoter and no transcription initiation complex is formed

Question 32

post transcriptional control: what happens during 5' capping?

Answer 32

- addition of a 5' cap, consisting of a modified form of guanine nucleotide at the 5' end of the primary RNA transcript - the 5' end of the pre-mRNA is capped as soon as it emerges from the RNA polymerase

Question 34

post transcriptional control: what happens during splicing?

Answer 34

- in RNA splicing - introns are cutout from pre-RNA and - exons flanking introns are spliced together (joined) - to form a mature mRNA with a continuous coding sequence

Question 35

what is the mechanism of RNA splicing?

Answer 35

1. RNA molecules known as **small nuclear RNAs (snRNAs)** are complexed with protein subunits to form **small nuclear ribonucleoproteins (snRNPs)** 2. snRNPs **associate with other proteins** to bind at the **splice sites** at each end of an intron to form a **spliceosome** 3. the spliceosome cut/**cleave**/excise at both ends of an intron to release the intron which is rapidly **degraded by nucleases** 4. at the same time, the **two exons flanking the intron are joined/ spliced** together by formation of phosphodiester bond

Question 36

what is alternative RNA splicing?

Answer 36

- excising/cleaving all introns or different number of introns from the same type of pre-mRNA - to generate many mature mRNA with diffferent combinations of exons - hence more than one type of polypeptides can be encoded by one gene - in alternative splicing, specific regulatory proteins contron intron/exon choices by binding to specific regulatory sequences within primary RNA transcript - this determines which region is to be used as splice sites

Question 37

what occurs during 3' POLY-A TAILING/ POLYADENYLATION

Answer 37

- the addition of adenine nucleotides at the 3' end of pre-mRNA

Question 38

what are the functions of 5' capping and poly-A tailing?

Answer 38

**maintain stability (increasing half-life) of the mRNA:** - 5' cap prevents the 5' end of the mRNA from being degraded by 5'-to- 3 exonuclease, an enzyme that cleaves nucleotides from the end (exo) of a polynucleotide chain. - The longer the poly (A) tail, the more stable the mRNA (longer-half-life of mRNA) hence longer duration for translation to occur, more protein synthesised. - Both structures may also be recognized by proteins which binds and prevent degradation. **Facilitate the transport of mature mRNA out of the nucleus:** - Proteins will bind to the 5' cap and the 3' poly-A tail and facilitate mRNA transport through the nuclear pore, out of the nucleus. **Facilitate ribosome attachment:** - once the mRNA reaches the cytoplasm both structures help ribosomes to bind and initiate translation.

Question 39

HALF LIFE (STABILITY) OF mRNA: **mechanism of RNA degradation: ** - 3'-to-5' ____ degrade the 3' poly-A tail of the mRNA until a ____ length. - this triggers the ____ enzymes that remove the 5' cap - once the 5' cap is removes, the mRNA is degraded by both ____ to____ and ____ to____ exonucleases Hence, the stability/ half-life of mRNA is hence determined by the length of the poly (A) tail. - The longer the poly (A) tail, the more ____ the mRNA (longer-half-life of mRNA) - hence longer duration for translation to occur, ____ proteins synthesised

Answer 39

**mechanism of RNA degradation: ** - 3'-to-5' **exonucleases** degrade the 3' poly-A tail of the mRNA until a **critical** length. - this triggers the **decapping** enzymes that remove the 5' cap - once the 5' cap is removes, the mRNA is degraded by both **5'** to **3'** and **3'** to **5'** exonucleases Hence, the stability/ half-life of mRNA is hence determined by the length of the poly (A) tail. - The longer the poly (A) tail, the more **stable** the mRNA (longer-half-life of mRNA) - hence longer duration for translation to occur, **more** proteins synthesised

Question 40

read! **Cytoplasmic addition of Poly (A) tail** - The initial polyadenylation of all eukaryotic mRNA occurs in the nucleus - In some cases, however, poly-A tails of specific mRNAs are lengthened by specific enzymes in the cytoplasun. - This prolongs the half-life/ stability of these specific mRNA, allowing increased duration of translation leading to more of these specific proteins produced.

Answer 40

lock in!

Question 41

what is the function and mechanism of Translational Repressor Proteins?

Answer 41

**Function**: - Translational repressor proteins regulate gene expression by blocking the initiation of translation for specific mRNAs. **Binding Site:** - These repressors bind to the 5′ Untranslated Region (5′ UTR) of the mRNA. - The 5′ UTR is a non-coding region upstream of the start codon (AUG). - It contains important regulatory sequences necessary for translation initiation. **Mechanism of Action:** - When the repressor protein binds to the 5′ UTR: - It physically blocks the ribosomal subunits (small and large) from assembling at the ribosome binding site near the 5′ end. - This prevents the ribosome from scanning or attaching properly to the mRNA, which is required to start translating the coding region. **Effect**: - The binding of the repressor results in no formation of the translation initiation complex, thus translation of the mRNA into protein is inhibited. **Regulatory Importance:** - This allows cells to control protein synthesis precisely, turning off production of certain proteins when they are not needed.

Question 42

**PROTEIN DEGRADATION VIA UBIQUITINATION:** - selective ubiquitination of proteins to limit the ____ of the protein - Adjust the kinds and ____ of its proteins in response to changes in its environment - Enables the cell to maintain its proteins in ____ working order - When proteins are ____/misfolded, they are usually broken down immediately and replaced by functioning ones.

Answer 42

**PROTEIN DEGRADATION VIA UBIQUITINATION:** - selective ubiquitination of proteins to limit the **lifespan** of the protein - Adjust the kinds and **amounts** of its proteins in response to changes in its environment - Enables the cell to maintain its proteins in **prime** working order - When proteins are **damaged**/misfolded, they are usually broken down immediately and replaced by functioning ones.

Question 43

describe the mechanism for protein degradation via ubiquitination:

Answer 43

1. To mark a protein for destruction, a small protein called **ubiquitin** binds to the protein 2. Giant protein complexes called **proteasomes** recognize the ubiquitin and isolate the **ubiquitinated protein in its central cavity.** 3. The enzymatic component of proteasomes **unfolds** and **degrades** the **ubiquitinated protein into short peptides** which can be further degraded by other enzymes in the cytosol. 4. The ubiquitin is released and can be recycled.