Control of Gene Expression & RNAi

Question 1

Why is control of gene expression important?

Answer 1

Structure & function of cell depends on what proteins are produced & in what quantities.
Differences in cell types & tissues come about by what gene is expressed & when.

Question 2

At what points in DNA > protein pathway can gene expression be controlled?

Answer 2

Transcriptional control
RNA processing control
RNA transport & localisation control
Translation control
mRNA degradation control
Protein activity control

Question 3

What set of genes does RNA Polymerase I transcribe?

Answer 3

5.8S, 18S & 28S rRNA genes

Question 4

What set of genes does RNA Polymerase II transcribe?

Answer 4

All protein coding genes, plus snoRNA, miRNA, siRNA, lncRNA & most snRNA genes.

Question 5

What set of genes does RNA Polymerase III transcribe?

Answer 5

tRNA, 5s rRNA, some snRNA & genes for other small RNAs

Question 6

How is gene expression controlled in the initiation of transcription?

Answer 6

Promoter required for RNA polymerase to bind & initiate transcription at target site
RNA polymerase interacts with other proteins (transcription factors) when it binds to promoter
Additional control sequences can determine when a gene is transcribed

Question 7

What are Cis-acting regulatory regions?

Answer 7

DNA sequences recognised by proteins.
Binding sites for one or more trans-acting proteins.
Present on the same molecule of DNA as the gene they regulate.

Question 8

What are Trans-acting proteins?

Answer 8

Transcription factors that bind to promoter & enhancer to control transcription from the gene.
Activators & repressors bind to enhancers.
General transcription factors & RNA pol. II bind to promoter.

Question 9

What are general transcription factors & their key roles?

Answer 9

Subunit of TFIID that is a TATA-box binding protein (TBP) binds to the TATA box
TBP recruits TFIID complex & TFIIB to promoter
RNA pol. II & further transcription factors are recruited to promoter
TFIIH promotes the opening of DNA & phosphorylation of RNA polymerase II (phosphorylation initiates transcription)

Question 10

Give the role of activator proteins.

Answer 10

Bound to enhancers upstream from promoter.

Interact with general transcription factors & RNA pol. II via a mediator complex

Question 11

Give an example of an activator protein.

Answer 11

Gal4 system of yeast
Gal4 protein is regulator for the transcription of multiple genes that are part of the galactose metabolism pathway
UASG -upstream activation sequence
In absence of galactose, when Gal4 binds to UASG, Gal80 protein bound to this that inhibits Gal4 no Gal1 gene is not transcribed.
When galactose is present, it binds to Gal80, releasing it from Gal4 and allowing it promote the transcription of Gal1 gene.

Question 12

What region of the DNA can fine-tune gene expression?

Answer 12

Complex regulatory regions.

Question 13

Give the mechanism that acts as a brake on runaway basal transcription.

Answer 13

Condensed chromatin.

Question 14

Give the mechanisms by which DNA is made accessible to transcription factors.

Answer 14

Nucleosome sliding allows access of transcription machinery to DNA.
Transcription machinery assembles on nucleosome-free DNA.
Histone variants allow greater access to nucleosome DNA.
Specific patterns of histone modification destabilize compact forms of chromatin & attract components of transcription machinery.

Question 15

How can core histones can be covalently modified on their N-terminal tails to force a transcription pattern at that site?

Answer 15

Histones can be covalently modified at many sites on their N-terminal tails
Acetylation, acetyl group added to lsyines, activates expression
Methylation, methyl groups added to lsyines or arginines, represses expression
Phosphate groups added to serines or threonines

Question 16

Give the histone H3 modifications that affect gene expression.

Answer 16

Acetylation
Methylation
Phosphorylation

Question 17

How can DNA be modified directly?

Answer 17

Cytosine can be methylated, typically occurs in vertebrates in the sequence CG, methylated DNA represses gene expression.
This pattern of cytosine methylation can be inherited after DNA replication. Example of epigenetics, maintains patterns of gene repression in differentiated cells.

Question 18

Can epigenetic mechanisms can contribute to gene repression?

Answer 18

Yes.

Question 19

How is gene expression different in bacteria compared to in eukaryotes?

Answer 19

No nuclear membrane, one cytoplasmic compartment, no histones, coupled transcription & translation in the cytoplasm.

Question 20

How is RNA polymerase recruited to the bacterial promoter?

Answer 20

Sigma subunit binds first, recruits rest of RNA polymerase

Different sigma factors bind to promoters of different sets of genes

Question 21

Describe bacterial gene expression.

Answer 21

Genes of related functions are clustered into OPERONS
An operon has a single promoter
All genes in a n operon are transcribed together
mRNA is translated to give separate proteins

Question 22

Give an example of bacterial gene expression.

Answer 22

Regulation of lac OPERON
Negative regulation; repressor molecule always produced, when lactose is present the repressor molecule is inhibited so transcription can occur.
-When lactose is absent a lac repressor protein is produced by lac I
lac repressor binds to lac0 site between promoter & lac OPERON
-When lactose is present, a metabolic by-product allactose binds to repressor molecule, so it cannot bind & inhibit lac0 region.

Question 23

Give the type of proteins involved in positive regulation.

Answer 23

Activator proteins.

Question 24

How can pre-mRNAs produce multiple products?

Answer 24

mRNA splicing
Intron inclusion or skipping
Alternative 5'/3' splice-site selection
Intron retention
Order DOES not change

Question 25

As alternative splicing can be tissue-specific, what do different splicing patterns depend on?

Answer 25

eg for tropomyosin RNA | Depends on the type of muscle that the protein is required for.

Question 26

How is alternative splicing actively regulated?

Answer 26

Negative control, repressor proteins bind to splice sites & mask them. Positive control, activator proteins bind in proximity of splice sites onto introns & attract the splicing machine.

Question 27

Give ways in which RNA editing changes sequence in mature mRNA and why it occurs.

Answer 27

Post-transcriptional editing of mRNA further increases the number of distinct proteins that can be encoded in a genome. Addition or deletion or uridine, deanimation of adenine to produce inosine, deanimation of cytosine to produce uracil.

Question 28

Give an example of how RNA editing changes sequence in mature mRNA.

Answer 28

Apo-lipoprotein B product is 4563 codons in liver & 2152 in intestine, because a C>U generates a stop codon in the longer sequence to form the shorter intestinal sequence.

Question 29

What affects the half life of mRNA?

Answer 29

Its sequence.

Question 30

Give an example of how UTR sequences can be in important in controlling mRNA lifetimes.

Answer 30

Ion regulation in mammals (can be toxic at very high levels) at low iron levels -transferrin receptor is a protein involved in iron uptake into the cell, iron response elements are present in the mRNA for transferrin -iron response proteins can bind to these elements and prevent degradation of the mRNA on the 3’ end, making it more stable, more transferrin translated & more iron uptake into cell. at high iron levels -iron binds to iron response protein & inhibits it binding to the elements on mRNA, so mRNA less stable and degraded from 3’ end & less transferrin translated. Less iron uptake into cell.

Question 31

When & how was RNAi discovered?

Answer 31

In C.elegans by 998 by Andrew Fire & Craig Mello. - injected RNA for a gene that produced a protein involved in muscle function, had no effect on gene expression. - if they injected antisense RNA it had no effect on gene expression. - if they injected double-stranded RNA it reduced the expression of specific genes. It does this by reducing levels of mRNA for that gene, the RNA interference (RNAi) is sequence-specific.

Question 32

Give the mechanism that RNAi mediates.

Answer 32

Resistance to both endogenous parasitic & exogenous pathogenic nucleic acids. Regulates the expression of protein-coding genes.

Question 33

Give the RNAi gene silencing process.

Answer 33

Works on double stranded RNA, can come from different sources, gets processed, binds to argonaute protein that strips away one of the strands of RNA. - that RNA bound to the protein can then find a target mRNA to also bind to. - binds by base-pairing rules. - once the complex is formed, the mRNA either: is cleaved & destroyed; has repressed translation & eventually targeted for destruction; or the complex forms a heterochromatin on DNA from the target mRNA has been transcribed.

Question 34

Give the process of miRNA processing

Answer 34

- form short 80 nucleotide hairpin structures, in nucleus they undergo cropping where 5’ and 3’ end are cut off. - then undergoes dicing, one strand is degraded by argonaute protein & loop cleaved off - RNA & argonaute complex is called RISC (RNA-induced silencing complex), finds a mRNA match - if mRNA match is extensive, will undergo slicing & mRNA is rapidly degraded - if mRNA match is less extensive, mRNA is rapidly translational repression & eventually is degraded.

Question 35

Give the importance of miRNA regulation.

Answer 35

Shown to be key regulators of gene expression in many biological processes

Question 36

How is RNAi used as an experimental tool for specific gene ‘knockdown’?

Answer 36

Can do this by feeding or injecting the organism with double stranded RNA that corresponds to the sequence of your target gene.

Question 37

How is RNAi used to control infectious diseases?

Answer 37

Generate dsRNA corresponding to dsRNA viruses & inject or feed them to the shrimp.