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4.1 Molecular Biology > Control of Gene Expression > Flashcards

Control of Gene Expression Flashcards

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1
Q

How can you know a gene is being expressed?

A

You cannot assume agene is working until you have an active protein product

i.e. gene expression is not just transcription of a gene

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2
Q

Give an example of when a gene is transcripted but not expressed
(3)

A

The mutation of delta f508 gene in CF

This results in a protein which has a fairly active ion channel

However the proteinas gets stuck in the golgi apparatus on its way to the cell membrane

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3
Q

Why do we need to regulate gene expression?

A

All cells in the human body have the same genome

Yet liver cells are distinctly different from cardiac cells

Gene expression is regulated to allow for the differentiation of cells

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4
Q

What does the mRNA from any cell represent?

A

Represents only the genes that are active in that cell at that moment in time

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5
Q

Talk about the expression of genes in Prader-Willi syndrome, and what problems arise in diagnosis?
(4)

A

Prader willi syndrome is caused by mutation of genes on chromosome 15

The gene is found on both copies of chromosome (maternal and paternal) but it is only expressed on the paternal gene

Therefore if you have paternal deletion you will have the syndrome -> you still have the gene but its not active

This can cause problems in detecting the syndrome as regular PCR will still detect the gene -> we need to look at the RNA instead to detect the syndrome

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6
Q

What is one reason why you might have transcription but no translation of a protein

A

mRNA can be attacked by miRNA (micrornase)

micro-RNAses can cause degradation of rna or failure to translate the rna

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7
Q

What are the basic steps in gene expression

A

DNA
Transcription by RNA polymerase II
hnRNA
Post transcriptional processing
mRNA
miRNAs and siRNAs
Translation
Protein
Post-translational modification
Protein product

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8
Q

What are three examples of post transcriptional processing?

A

Capping
Processing
Polyadenylation

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9
Q

Give two examples of RNases that act on mRNA

A

miRNAs
siRNAs

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10
Q

What are siRNAs?

A

Small interfering RNA

Also known as short interfering RNA or silencing RNA

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11
Q

What is capping?
(3)

A

The enzymatic modification of the 5’ end of mRNA

This protects the strand from degradation

A 7-methylguanosine (modified guanine) is added to thr 5’ end

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12
Q

What is polyadenylation?

A

The addition of a string of As

Addition of a poly (A) tail to the 3’ end

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13
Q

What is splicing?

A

The removal of any introns

Happens after transcription

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14
Q

What is RNA called before it has undergone post transcriptional processinf?

A

hnRNA or heterogeneous nuclear RNA

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15
Q

What is hnRNA?

A

Heterogeneous nuclear RNA

Is an immature form of mRNA which has yet to undergo post transccriptional processing such as splingin, capping and polyadenylation

i.e. it still contains introns and is not protected from degradation

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16
Q

What is the point of post transcriptional processing?

A

To make a chain of only exons

Which is protected from degradation

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17
Q

What signals the gene exoression?

A

Extracellular signals switch on genes

This then signals decompaction of DNA for transcription to occur

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18
Q

What enzyme is responsible for unwinding DNA?

A

Helicase enzyme

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19
Q

What is the role of RNA polymerase II

A

This transcribes HnRNA

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20
Q

What are the different levels of control over gene expression?

A

ALteration of chromatin structure
Epigenetic
Initiation of transcription by RNA pol II
Post transcriptional processing
Transport to cytoplasm
mRNA stability and degradation
Translation of RNA at ribosome
Post translational processing

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21
Q

How is gene expression regulated at a chromatin level
(3)

A

Most of DNA in a cell is highly compacted

In this form DNA is not available for transcription as helicase enzymes cannot get near DNA etc

The gene has to be made available for transcription

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22
Q

How is gene expression regulated on an epigenetic level?
(5)

A

Associated with chemical modifcation of DNA and of the proteins associated with DNA

Methylation especially of the cpg island

methylation of DNA impairs transcription

Genes not in use tend to be methylated

Abberant methylation of incorrect genes and acetylation of incorrect genes seen in cancer

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23
Q

Talk about the role of miRNAse in gene regulation

A

30% of human genes are regulated by micRNAse

Cancers tend to have disregulation of micRNAse

These bind to mRNa and either prevent it from being translated or they degrade it -> either ay we dont get a protein

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24
Q

How is gene expression controlled on a translational level

A

mRNA is translated to a protein which is often times not active -> protein needs activation

Transport, phosphorylation, glucosylation are all examples of post-translational processing needed to activate a protein

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25
Q

How is gene expression controlled on a translational level

A

mRNA is translated to a protein which is often times not active -> protein needs activation

Transport, phosphorylation, glucosylation are all examples of post-translational processing needed to activate a protein

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26
Q

What are the three differnet types of control on gene expression?

A

Developmental

Tissue specific

Environmental

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27
Q

What is meant by developmental contro over gene expression?

A

Temporal gene regulation

Gene regulation necessary for us to develop i.e. how do we get from a single cell to a human

Involves the switching on of genes to allow for cell differentiation and tissue development etc

Genes turned on due to a developmental need

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28
Q

What is meant by spatial control over gene regulation

A

Growth hormone produced only by cells in the pituitary gland

What signals are in place to control this -> the signals that only exist in these cells in this organ

What signals cause abberent expressio in disease states

Genes turned on due to their location

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29
Q

What is meant by environmental control of gene expression?

A

If you ingest heavy metals -> metallothionine genes switch on to deal with the heavy metals

i.e. genes only turned on because of external environmental factors

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30
Q

What are the different levels of chromatin structure?
(5)

A

Condensed scaffold-associated form

Extended scaffold-associated form

30nm chromatin fiber of packed nucleosomes

‘Beads-on-a-string’ form of chromatin

Short region of DNA double helix

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31
Q

At what chromatin levels is transcription possible, i.e. at what levels of unraveling can the helicase enzyme work

A

‘Beads-on-astring- form of chromatin

Short region of DNA double helix

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32
Q

What is meant by the ‘bead-on-a-string’ structure?

A

Nucleosomes

An optimer of histone (8 histones brought together)

DNA wraps around each histone to create the bead on a string shape

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33
Q

What forms of chromatin are you most likely to see in the cell?

A

Very rarely see chromosome in metaphase (traditional look of a chromosoem you imagine) -> very little gene expression possible in this form so only seen when cell is dividing

30nanometer chromatin fibre seen much more commonly but transcription still not possible in this form -> vast majority in this form

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34
Q

Talk about gene expression in Huntingtons disease

A

Huntingtons disease is a dominant disease - only need one copy of mutated chromosome

Mutation of chromosome 4

If you inherit one copy of the mutated gene you will suffer from the disease as the gene is pathogenic

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35
Q

Explain why some genetics are recessive and others are dominant

A

Mutations in dominant conditions tend to be pathogenic

Mutations in recessive conditions tend to be loss of function mutations -> hence why two copies of the mutation are required for the gene as you will still produce some functional protein

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36
Q

Give two examples of X linked disorders

A

Fragile X syndrome

Duchenn muscular dystrophy

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37
Q

Explain why X linked disorders affect only males

A

Males only have one copy of X chromosome while females have two

Males get full affect of any mutation on X chromosome while females can just use their second copy

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38
Q

What happens in expression in Downs Syndrome?
(3)

A

Extra opy of chromosome 21

Symptoms occus due to too much protein from genes on chromosome 21

Genes in a certain region of 21 are directly responsible

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39
Q

What is epigenetics?
(4)

A

It means above or in addition to genetic

Non-sequence dependant inheritence

Responsible for dfferentiation i.e. makes certain genes available for transcription

The reason why identical twins can still be different

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40
Q

Give an example of where epigenetics is important
(2)

A

When a muscle stem cell divides it will only ever make muscle cells even though muscle stem cells are similar to other stem cells

Every one of these cells have the same DNA so how do these cells know what genes should be active -> epigenetics

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41
Q

Give an example of where epigenetics is important
(2)

A

When a muscle stem cell divides it will only ever make muscle cells even though muscle stem cells are similar to other stem cells

Every one of these cells have the same DNA so how do these cells know what genes should be active -> epigenetics

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42
Q

Give some examples of the work of epigenetics
(3)

A

Identical twins with different natural hair colour

A single individual with two different eye colours

An identical twin liter mate with different coat colours

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43
Q

What was the Duke University carried out on clone mice to prove the effects of epigenetics?
(4)

A

Clone mice with identical genomes

Blastocysts were implanted into two different mice mothers

The mice mothers were fed different diets

The mice babies came out with completely different appearances

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44
Q

In what four ways are epigenetics controlled?

A

Chromatin structure - compacted vs uncompacted

Chemical modification of chromatic - methylation

Alternative splicing of RNA

RNA interference via miRNA and siRNA

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45
Q

Explain how we contol genetics through methylation

A

Via acetylation of histones and methylation of histones

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46
Q

Why is there a need for alternative splicing?
(3)

A

Most genes in the human genome produce more than one protein, because of this they dont need to use all exons everytime the gene is transcribed i.e. they only need some

e.g. Might use exon 1, 2, 3, and 4 for one protein but then might use exon 2, 3, and 5 for another protein

Its still the sam gene but can just have different products e.g. DMD gene

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47
Q

Why is there a need for alternative splicing?
(3)

A

Most genes in the human genome produce more than one protein, because of this they dont need to use all exons everytime the gene is transcribed i.e. they only need some

e.g. Might use exon 1, 2, 3, and 4 for one protein but then might use exon 2, 3, and 5 for another protein

Its still the sam gene but can just have different products

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48
Q

What does the work of miRNA or siRNA do?

A

It results in protein product

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49
Q

What does the work of miRNA or siRNA do?

A

It results in protein product

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50
Q

How does methylation control DNA transcription in an active cell vs an inactive cell?

A

Active cell:
- Open, active chromatin tends to be unmethylated (especially at promoter)
- The Histones of the chromatin, particularly H3 tend to be acetylated

Inactive cell
- Condensed chromatin, chromatin methylated (including at promoter)
- deacetylated/unacetylated histones

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51
Q

Talk about methylation of cytosines as a form of gene control

A

Sequence of Cs and Gs knon as CPG island

Cs and Gs are subject to methylation which inactivates DNA

Cs and Gs on both sides of strand are methylated

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52
Q

Give an example of a condition whereby CpG methylation is important

A

In fragile X the FMR1 gene is mutated to repeat CCGs

Mutations of over 200 CCG repeats results in methylation of the promoter and thus failure of the gene

This causes male intellectual disability

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53
Q

Give a basic description of the structure of histones, how do they compact DNA

A

A histone is a protein that provides strucutural support for DNA double helix

2 copies of histone A, B, C and D come together to make an optimer i.e. 8 histones make an optimer

DNA double helix wraps around the histone optimer

Each histone has a tail which extends out past the DNA double helix

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54
Q

How are histone molecules involved in gene control

A

Modification of the tails, particuarly H3 result in activation/inactivation of DNA -> H4 is often involved as well

H3 Lysine 4 methylation results in activation

H3 Lysine 9 methylation results in chromatin condensation which is associated with transcriptional repression

Acetylation of Lysine 9 activates transcription

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55
Q

How is H4 involved in gene controll?

A

Methylation of lysine 16 has transcriptional activation

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56
Q

How is H4 involved in gene controll?

A

Methylation of lysine 16 has transcriptional activation

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57
Q

What percentage of the human genome is regulated by methylation, how do we know this?
(3)

A

If a gene has a CPG island in its promoter it is controlled by methylation -> think of H3 and H4

60% of genes are have CPG islands

The remaining 40% have TATA sequences

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58
Q

How can methylation prevent transcription?

A

Methylation can prevent trascription complex from binding to a downstream to a promoter

i.e. if CpG island is methylated upstream it can prevent transcription complex from binding to a TATTA sequence downstream
-> this is because methylation of CpG is associated with chromatin condensation

??? im not sure on this

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59
Q

Why do genes require a promoter sequence such as GC or TATTA?

A

This is where RNA polymerase II will bind

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60
Q

What does an enhancer sequnce do and where are they found?

A

These modulate the rate of transcription

They are found throughout a sequence even in introns

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61
Q

What gene in the human genome has the most exons?

A

The Titin gene (TTN)

It has over 363 exons

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62
Q

Most conditions are caused by mutations in exons, give an example of a conditions whereby there is mutations within introns?

A

In Friedreichs ataxia

There is a mutation in intron 1 of the FXN gene

This results in expansion of a GAA-TC repeat tract which leadds to an mRNA deficit

Failure to transcribe and make mRNA

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63
Q

What are the four core promoter elements of non CpG island promoters?

A

BRE
TATA box
Inr/initiator
DPE/ downstream core promoter element

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64
Q

What are the four core promoter elements of non CpG island promoters?

A

BRE
TATA box
Inr/initiator
DPE/ downstream core promoter element

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65
Q

What is BRE?

A

TFIIB recognition element

Its found commonly enough and found near to the TATA sequence

GGG/CCA followed by CGCC

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66
Q

What is the sequence of a TATA box

A

There is some variability:

TATA followed by (A/T)A(A/T)(A/G)

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67
Q

What is the Inr?

A

An initator sequence

There is lots of variability in this sequence

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68
Q

What is DPE?

A

Downstream core promoter element

Usually found in the first exon

Lots of variation again in this sequence

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69
Q

What was the first hypothesis put forward on promoter sequences?

A

That the 5’ flanking sequence contributes to the initiation of transcription of genes

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70
Q

Give four examples of promoter sequences

A

TATA box
CAAT box
GC box
Oct site

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71
Q

Talk a little about the variation that occurs in the TATA box

A

There is some variation in all bases but its usually TATA followed by some sequence of Ts and As

100% of the time there is a T in the 3rd position

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72
Q

What is an example of a model promoter that we used to prove TATA was involed in transcription?

A

AMLP
Adenovirus major late promoter

But any TATAA containing sequence could be used

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73
Q

Give three examples of reporter genes we could use to prove TATA was involed in transcription?

A

Chloramphenicol acetyl transferase (CAT)

Luciferase

B Galactosidase

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74
Q

How to prove TATA was involed in transcription?

A

We connected TATAA to a gene whos product is easy to detect and put it into a cell that doesnt usually produce this produce

We then detected either the RNA or the product protein to prove our hypothesis

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75
Q

How does the CAT/Chloramphenicol acetyl transferase assay work to prove TATA was involed in transcription?
(4

A

We used the gene for chloramphenicol

We used promoters specific for chloramphenicol

Chloramphenicol protein produced

We then converted this to chloramphenicol acetyltransferase for easy detection

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76
Q

How does the B Galactosidase reaction work?

A

It uses the same method to prove TATAA through CAT except it produces light which is measured

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77
Q

How does the B Galactosidase reaction work?

A

It uses the same method to prove TATAA through CAT except it produces light which is measured colourimetrically

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78
Q

How does the luciferase assay work to prove TATA

A

It produces light which is measured

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79
Q

How does the luciferase assay work to prove TATA

A

It produces light which is measured

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80
Q

When using a bacterial plasmid to make a eukaryotic protein, what must be added into the plasmid?

A

You must add in a synthetic poly(A) signal to ensure the polyadenylation of the gene

Mammalian genes are polyadenylated while bacterial genes are not -> gene will not be translated if not polyadenylated

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81
Q

How would you detect the RNA of the CAT asay?

A

Reverse-trannscription PCR

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82
Q

What are the steps of reverse Pcr

A

Transfect construct

Isolate mRNA from transfected cells

Reverse transcribe RNA using reverse transcriptase

This gives us cDNA (for all genes)

PCR amplify using specific primers for CAT this cDNA to give us multiply copies

If we get a product then our assay has worked - Very high sensitivity

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83
Q

What are the steps in a northern blot?

A

Northern Blot

Transfect construct

Isolate RNA from transfected cells

Run on agarose gel first

Transfer to nitrocellulose membrane

Apply/Probe membrane with 32P labelled CAT probe

Detect presence /absence of signal/radioactive probe by applying an xray film and then developing it

Low Sensitivity

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84
Q

What are the steps in a northern blot?

A

Northern Blot

Transfect construct

Isolate RNA from transfected cells

Run on agarose gel first

Transfer to nitrocellulose membrane

Apply/Probe membrane with 32P labelled CAT probe

Detect presence /absence of signal/radioactive probe by applying an xray film and then developing it

Low Sensitivity

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85
Q

What are the steps to the nuclease protection assay

A

Transfect cell line with reporter construct

Isolate RNA from transfected cells (single stranded)

Hybridise with radiolabelled CAT probe -> forms double strands where binding occurs

Treat hybridisation mix with Sl nuclease -> degrades all single stranded molecules

Electrophorese and autoradiograph

Only gene of interest remains

Medium Sensitivity

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86
Q

What are the steps to the nuclease protection assay

A

Transfect cell line with reporter construct

Isolate RNA from transfected cells (single stranded)

Hybridise with radiolabelled CAT probe -> forms double strands where binding occurs

Treat hybridisation mix with Sl nuclease -> degrades all single stranded molecules

Electrophorese and autoradiograph

Only gene of interest remains

Medium Sensitivity

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87
Q

What is the principle behind the CAT assay?

A

Chloramphenicol + acetyl coenzyme A

Produces acetyl chloramphenicol

Acetyl chloramphenicol runs to a separate spot on gel than chloramphenicol

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88
Q

What is the principle behind the CAT assay?

A

Chloramphenicol + acetyl coenzyme A

Produces acetyl chloramphenicol

Acetyl chloramphenicol runs to a separate spot on gel than chloramphenicol

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89
Q

What are the main steps of the CAT assay

A

Chlorampheincol Acetyltransferase
Assay (CAT Assay)
Transfect construct
Incubate —48 hours
Isolate cytoplasmic extract
Incubate extract with 14C-Chloramphenicol and Acetyl
Coenzyme A
Extract Chloramphenicol products
Run Thin Layer Chromatography (TLC) to separate
14C- chloramphenicol and acetyl 14C-chloramphenicol
Autoradiograph

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90
Q

Why is the CAT assay considered a good method of assay

A

Visually very good at visualising levels of expression

Can clearly see between mono and diacetylated Chloramphenicol

If there is really high expression you might even see a triacetylate

  • dont do this assay anymore though
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91
Q

What kind of CAT assay do we run now?

A

CAT sandwich ELISA

Use a plate coated with anti-CAT antibody

Blue colour

ELISA plate reader used

Nice simple assay which is also quantitative and results expressed graphically

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92
Q

Why do we often use the luciferase assay vector?

A

One of the quickest reporter assays

Luciferase RNA or enzyme product can be detected

Tube will immediately glow

Use a luminometer to read level of lfuorescence to determine amount f product

So much quicker and also quantitative

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93
Q

How does the b-galactosidase reporter assay work, why do we use it?

A

We use it because it is the cheapest method

It results in blue coloured cells which are read microscopically

It can be used as a stain -> cell/animals will be blue if they have been able to express the gene

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94
Q

How can B-galactosidase be used to prove expression of genes?

A

Used like a dye

Promoter connected to B-galactosidase

Expression of gene results in expression of blue dye

Promotive active in active genes

used in embryos - to display where certain genes are expressed etc

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95
Q

What is the TATAA box required for?

A

Transcription

The TATAA box is a DNA sequence essential for the initiation of transcription in eukaryotic cells.

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96
Q

What role does TATTAA play in the transcription process?

A

Acts in an orientational position

TATTAA helps position the transcription machinery correctly for effective transcription.

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97
Q

What happens to the catalytic subunit without TATAA?

A

It won’t bind correctly

The correct binding of the catalytic subunit is crucial for transcription initiation.

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98
Q

How does the positioning of the TATA box affect transcription?

A

Orientation and position dependent

The TATA box must be in a specific position relative to the gene start site to function properly.

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99
Q

What experimental setups were used to study the TATA box?

A

Reporter constructs with varied TATA box positions and orientations

These experiments included moving the TATA box closer to or further from the start site, placing it in incorrect orientations, or mutating it.

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100
Q

What was concluded from the alteration of the TATA box in experiments?

A

Interferes with transcription

Changes to the TATA box’s position or orientation negatively impact the transcription process.

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101
Q

Fill in the blank: The TATA box must be in a _______ position with respect to the start of the gene to function.

A

defined

A defined position is essential for the TATA box to effectively facilitate transcription.

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102
Q

What is the main focus of the experiment shown in Slide 59?

A

The effects of individual base mutations on transcription

The experiment involved generating 100 CAT reporter constructs with mutant promoters and analyzing RNA or CAT protein after transfection into cells.

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103
Q

What is the relationship between conserved sequences and transcription?

A

Mutations in conserved sequences tend to interfere with transcription

This indicates that conserved sequences play a critical role in the transcription process.

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104
Q

How many CAT reporter constructs were generated for the experiment?

A

100 CAT reporter constructs

Each construct had different mutant promoters.

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105
Q

What was observed when certain bases that bind NF1 were mutated?

A

A severe deficit in transcription

NF1 is a transcription factor, and its binding is crucial for transcription initiation.

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106
Q

What was the outcome of some mutations in certain regions regarding transcription?

A

Some mutations actually improved transcription

This highlights that not all mutations are detrimental; some may enhance transcriptional activity.

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107
Q

Fill in the blank: Mutations in _______ sequences have less of an effect on transcription.

A

non-conserved

Non-conserved sequences do not play as significant a role in transcription regulation.

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108
Q

True or False: All mutations in conserved sequences lead to improved transcription.

A

False

While some mutations may improve transcription, most tend to interfere with it.

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109
Q

What method was used to analyze the effects of the mutations on transcription?

A

Analysis of RNA or CAT protein

This analysis was performed after transfections into cultured cells.

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110
Q

What is absolutely required for transcription?

A

Promoter

The promoter region is essential for initiating the transcription process in molecular biology.

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111
Q

What is the function of the TATA box?

A

Acts as a core promoter in an orientation and position dependent manner

The TATA box is a crucial element in the promoter region that helps in the binding of RNA polymerase.

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112
Q

What happens to transcription if there are mutations in the TATA box?

A

Almost always completely abolishes transcription

Mutations can disrupt the binding of transcription factors and RNA polymerase.

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113
Q

What must be kept constant between the TATA box and the start site of transcription?

A

Sequence length

The specific sequence itself is unimportant, but the length must remain consistent for proper transcription initiation.

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114
Q

Which non-core promoter elements can be important for transcription?

A

CAAT, GC

These elements can enhance or regulate the transcription process, though they are not part of the core promoter.

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115
Q

True or False: The TATA box can be moved or flipped in orientation.

A

False

The TATA box’s position and orientation are critical for its function as a core promoter.

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116
Q

What is the primary function of metallothionein genes?

A

These respond to heavy metal ingestion

Metallothioneins are involved in the detoxification of heavy metals.

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117
Q

What key element is found in the promoter region of the metallothionein gene?

A

TATA box

The TATA box is essential for the initiation of transcription.

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118
Q

What type of region do metallothionein genes have in addition to the TATA box?

A

GC rich region

GC rich regions are often involved in the regulation of gene expression.

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119
Q

Which protein is indicated to bind to the GRE in the metallothionein promoter?

A

Steroid receptor

The steroid receptor is involved in the response to glucocorticoids.

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120
Q

What are the elements that contribute to metal induction in the metallothionein promoter?

A

MRE (Metal Response Elements)

MREs are specific sequences that facilitate the binding of metal-responsive proteins.

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121
Q

Fill in the blank: The regulatory region of a human metallothionein gene contains _______ elements in both its promoter and enhancer.

A

regulatory

Regulatory elements are crucial for gene expression control.

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122
Q

Which transcription factors are indicated to bind to the metallothionein enhancer?

A

AP2 and AP1

AP2 and AP1 are involved in the regulation of gene expression in response to various signals.

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123
Q

What is the role of the Methionine response sequence in the metallothionein gene?

A

Involved in response to methionine levels

Methionine response sequences help regulate the expression of genes based on methionine availability.

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124
Q

True or False: The metallothionein promoter contains elements for both metal induction and glucocorticoid response.

A

True

This dual responsiveness allows for fine-tuned regulation of metallothionein expression.

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125
Q

What does the presence of multiple binding sites in the metallothionein promoter indicate?

A

Regulatory complexity

Multiple binding sites allow for the integration of various signaling pathways.

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126
Q

What is the start site for transcription?

A

The start site is typically characterized by sequences such as Oct, GC, CAAT, TATAA, and INR

127
Q

What are the components of a gene structure in the context of transcription?

A

The components include:
* Intron
* Exon 1
* Exon 2
* Intron 2
* Poly-adenylation signal
* Exon 3

128
Q

What sequence do proteins bind to during transcription initiation?

A

Proteins bind to the TATA box sequence

129
Q

How must proteins bind to the TATA box?

A

Proteins must bind in an orientation-specific manner

130
Q

What does RNA polymerase II transcribe DNA into?

A

RNA polymerase II transcribes DNA into heterogeneous nuclear RNA (hnRNA)

131
Q

True or False: RNA polymerase II can transcribe DNA from RNA.

A

False

132
Q

Fill in the blank: A hypothesis is that a protein or a complex of proteins is binding to the _______.

A

TATA sequence

133
Q

What is a logical hypothesis regarding RNA polymerase II and the TATA box?

A

RNA polymerase II is binding to the TATA box

134
Q

What aspect of protein binding to the TATA box is crucial for transcription initiation?

A

Binding must be orientation and position dependent

135
Q

What is the first step in proving the hypothesis that RNA polymerase II binds to the TATA box?

A

Designing an experiment to demonstrate the binding

136
Q

What does RNA polymerase 1 transcribe?

A

rRNA

RNA polymerase 1 is essential for synthesizing ribosomal RNA, which is crucial for protein synthesis in cells.

137
Q

What is the function of RNA polymerase 2?

A

Transcribes mRNA

RNA polymerase 2 plays a key role in synthesizing messenger RNA, which carries genetic information from DNA to ribosomes.

138
Q

What does RNA polymerase 3 transcribe?

A

tRNA and small nuclear RNAs

RNA polymerase 3 is involved in the transcription of transfer RNA and small nuclear RNAs, important for protein synthesis and RNA processing.

139
Q

What percentage of RNA in a cell is made up of rRNA?

A

16%

Ribosomal RNA is a significant component of the cellular RNA pool, playing a critical role in protein synthesis.

140
Q

True or False: Regulation of rRNA transcription is as stringent as that of mRNA.

A

False

The regulation of rRNA transcription is not as strict as mRNA regulation, which is crucial for determining cell phenotype.

141
Q

Fill in the blank: RNA polymerase 1 makes _______.

A

Ribosomal RNA

Ribosomal RNA is necessary in every cell for protein synthesis.

142
Q

What is RNA polymerase II?

A

A multisubunit complex (~14 subunits) that transcribes DNA into hnRNA

hnRNA stands for heterogeneous nuclear RNA, which is the primary transcript in eukaryotic cells.

143
Q

Can RNA polymerase II bind DNA independently?

A

No, it requires other factors (proteins) to facilitate DNA binding and transcription

This indicates that RNA polymerase II is not sufficient on its own for transcription.

144
Q

What is the function of the L’ subunit of RNA polymerase II?

A

It has a carboxy terminal domain (CTD) repeat of 7 amino acids that can be phosphorylated

The CTD is critical for the regulation of transcription and processing of the RNA transcript.

145
Q

What is the amino acid sequence of the CTD repeat in the L’ subunit?

A

Tyr-Ser-Pro-Thr-Ser-Pro-Ser

This sequence is repeated many times and is involved in phosphorylation during transcription.

146
Q

How many subunits does RNA polymerase II have?

A

Approximately 14 subunits

This multi-subunit structure contributes to its complex functionality in transcription.

147
Q

Fill in the blank: RNA polymerase II transcribes DNA into _______.

A

hnRNA

hnRNA is the precursor to mRNA in eukaryotic cells.

148
Q

True or False: The phosphorylation of the CTD repeats in RNA polymerase II is important for transcription.

A

True

Phosphorylation of the CTD is a key regulatory mechanism in the transcription process.

149
Q

What happens if you mix RNA polymerase II with a sequence containing TATA?

A

It won’t bind without additional factors

The TATA box is a DNA sequence that indicates where a genetic sequence can be read and transcribed, but RNA polymerase II cannot bind to it alone.

150
Q

What is the role of TFIID in transcription initiation?

A

TFIID is the first fraction identified that binds to DNA and helps in transcription initiation

TFIID is a complex that includes the TATA-binding protein (TBP) and is essential for the assembly of the transcription machinery.

151
Q

Which transcription factor is necessary for RNA polymerase binding to TATA?

A

TFIIE

TFIIE is crucial for the initiation of transcription as it allows RNA polymerase to bind to the TATA box.

152
Q

What is the function of TFIIA in the transcription process?

A

TFIIA aids in stabilizing the binding of TFIID to the TATA box but does not facilitate RNA polymerase binding on its own

TFIIA enhances the interaction between TFIID and DNA.

153
Q

Which transcription factor is identified after TFIID and TFIIA that allows RNA polymerase to bind?

A

TFIIB

TFIIB is essential for the recruitment of RNA polymerase II to the promoter.

154
Q

What does the TATAA box signify in a typical gene?

A

The TATAA box is a core promoter element that is crucial for transcription initiation

It is located approximately 25-30 base pairs upstream of the transcription start site.

155
Q

Fill in the blank: The transcription initiation complex is built up of TFIID, TFIIA, TFIIB, and _______.

A

TFIIE

TFIIE is the final factor required for the initiation of transcription.

156
Q

True or False: RNA polymerase binds to TATA without any additional transcription factors.

A

False

RNA polymerase requires the presence of transcription factors like TFIID, TFIIA, TFIIB, and TFIIE to bind to the TATA box.

157
Q

What happens when RNA polymerase is added after TFIIB binds?

A

RNA polymerase binds, but transcription does not start until TFIIE is added

This indicates that TFIIE is necessary for the initiation of transcription.

158
Q

What experimental approach was used to identify the transcription factors binding to DNA?

A

Making a nuclear extract, fractionating it, and testing fractions for DNA binding

This method allowed researchers to sequentially identify the transcription factors involved in the initiation complex.

159
Q

What is the significance of the experimental finding that purified RNA alone does not bind to TATA?

A

It indicates that additional factors are required for RNA binding

This highlights the importance of transcription factors in the transcription process.

160
Q

List the transcription factors involved in the transcription initiation complex in the order they were identified.

A
  • TFIID
  • TFIIA
  • TFIIB
  • TFIIE

These factors work sequentially to form the complete transcription initiation complex, allowing RNA polymerase to initiate transcription.

161
Q

What is the role of TFII30 in molecular biology?

A

TFII30 is an inhibitor that causes non-functional binding.

162
Q

What must be added for the transcription process to start?

A

TEIIA must be added.

163
Q

What does TFIIB do in the transcription process?

A

TFIIB recruits RNA polymerase II and provides structure for it to work from.

164
Q

List the order of transcription factors involved after TFIIB.

A
  • TFIIF
  • TFIIE
  • TFIIH
165
Q

True or False: TFIIB functions independently without other transcription factors.

A

False

166
Q

Fill in the blank: TFIIB recruits _______.

A

[RNA polymerase II]

167
Q

What is the significance of TEIIA in the transcription process?

A

TEIIA is necessary for the transcription process to initiate.

168
Q

What is TFIID composed of?

A

TBP and approximately 10 TAFs

Total size is around 800kDa

169
Q

What does TBP recognize and bind to?

A

TATA box sequence

170
Q

In which groove of DNA does TBP bind?

A

Minor groove

171
Q

What effect does TBP binding have on the DNA strand?

A

Results in the bending of the DNA strand

172
Q

What can inhibit TBP binding?

A

TF_230

173
Q

True or False: TBP binds to the major groove of DNA.

A

False

174
Q

What is the role of TFIIA in relation to TBP?

A

May bind DNA in conjunction with TBP

175
Q

What is the size of TBP?

A

Approximately 30kDa

176
Q

What is the total size of TFIID?

A

Approximately 800kDa

177
Q

Fill in the blank: TBP binds to the _____ groove of DNA.

A

minor

178
Q

What is suggested about TFIIA’s relationship with TFIIB?

A

It is quite possible that TFIIA is part of TFIIB

179
Q

What is the significance of TBP binding to the minor groove?

A

It probably helps the transcription process

180
Q

What is the role of TFIIA in transcription?

A

Binds to TFIID, possibly in solution and may activate TBP by relieving the repression caused by TFTF

TFIIA binds upstream of TFIID to facilitate transcription initiation.

181
Q

What does TFIIB interact with?

A

Interacts with TFID and TFIIF

TFIIB binds upstream of TBP to a BRE sequence in some promoters.

182
Q

Where does TFIIB bind in relation to TBP?

A

Binds upstream of TBP to a BRE sequence and downstream to the minor groove of DNA

This positioning may help align the transcription complex correctly.

183
Q

What is the main role of TFIIB?

A

To recruit RNA polymerase II

TFIIB acts as a bridge between TFIID and RNA polymerase II.

184
Q

How does TFIIB contribute to the transcription process?

A

Provides a surface (domain) that is recognized by RNA polymerase II

This interaction is crucial for the assembly of the transcription complex.

185
Q

True or False: TFIIB can allow transcription to occur on its own.

A

False

TFIIB does not initiate transcription by itself; it requires other factors.

186
Q

TFIIB is known to interact with which other transcription factors?

A

D and F

These interactions help in aligning the transcription process.

187
Q

Fill in the blank: TFIIB binds upstream from TFIID from the start site of transcription and probably aligns the whole _______.

A

transcription process

This alignment is essential for proper transcription initiation.

188
Q

What is the function of TFIIB in relation to the transcription complex?

A

Acts as a bridge between TFIID and RNA polymerase II

This bridging is crucial for the assembly and stability of the transcription complex.

189
Q

What is the composition of TFIIF?

A

Composed of 4 subunits

Includes 2 X RAP 74 and 2 X RAP 30

190
Q

What is one of the functions of TFIIF?

A

Interacts with CTD phosphatase

CTD refers to the C-terminal domain of RNA polymerase II

191
Q

How does TFIIF interact with DNA?

A

Interacts with DNA on either side of the TATA box

TATA box is a core promoter element

192
Q

What role does TFIIF play in relation to RNA Polymerase I?

A

May bind to and bring RNA Polymerase I to the transcription complex

193
Q

What complex does TFIIF stabilize?

A

Stabilises TBP - DNA – TFIIB complex

194
Q

What is a requirement for elongation in transcription?

A

Required for elongation

195
Q

Which transcription factors does TFIIF facilitate binding with?

A

Required for TFIIE and TFIIH binding

196
Q

What is the composition of TFIIE?

A

Heterotetramer (2 X 34kDa, 2 X 56kDa)

197
Q

To which molecule does TFIIE bind directly?

A

Binds directly to RNA polymerase II

198
Q

How does TFIIE interact with DNA?

A

Binds to DNA nonspecifically

199
Q

What is one of the roles of TFIIE in transcription?

A

Mediates assembly of TFIIH

200
Q

What possible activity does TFIIE have?

A

Possible helicase activity

201
Q

What does TFIIE do to the helicase activity of TFIIH?

A

Represses helicase activity of TFIIH

202
Q

What happens to TFIIE upon phosphorylation of the CTD?

A

Phosphorylation of CTD releases TFIIE and allows unwinding of DNA

203
Q

Fill in the blank: TFIIF is involved in bringing _______ to the transcription complex.

A

[RNA Polymerase I]

204
Q

TFIIF stays on during which phase of transcription?

A

Elongation phase

205
Q

What is the major role of TFIIF?

A

Recruit H

206
Q

True or False: TFIIF definitely has helicase activity.

A

False

207
Q

What is the effect of CTD tail phosphorylation on TFIIE?

A

Releases TFIIE

208
Q

What is the primary function of TFIIH in molecular biology?

A

TFIIH has DNA repair activity and phosphorylates the CTD tail of RNA polymerase II.

TFIIH is a multi-subunit complex involved in transcription and nucleotide excision repair.

209
Q

What activities are associated with TFIIH?

A

TFIIH is associated with the following activities:
* ATPase activity
* Helicase activity
* Kinase activity

These activities are crucial for its role in transcription and DNA repair.

210
Q

What does kinase activity refer to?

A

Kinase activity refers to phosphorylation activity.

Kinases are enzymes that catalyze the transfer of phosphate groups.

211
Q

What happens to the CTD tail of RNA polymerase II upon phosphorylation?

A

Phosphorylation leads to the release of the unphosphorylated CTD tail from TBP and initiates transcription elongation.

TBP stands for TATA-binding protein, which is part of the transcription initiation complex.

212
Q

Fill in the blank: The unphosphorylated CTD tail of RNA polymerase II is bound to _______.

A

[TATA box protein]

213
Q

True or False: Phosphorylation of the CTD tail of RNA polymerase II is necessary for transcription to occur.

A

True

Phosphorylation of the CTD tail is a critical step in the transition from transcription initiation to elongation.

214
Q

How does the phosphorylation of the CTD tail affect transcription?

A

It leads to transcription elongation after release from TBP.

This process is essential for the synthesis of RNA from the DNA template.

215
Q

What is the state of RNA polymerase II when the initiation complex is built up?

A

Unphosphorylated

The unphosphorylated state of RNA polymerase II is essential for the formation of the initiation complex.

216
Q

Which transcription factor phosphorylates RNA polymerase II as a requirement for the elongation stage?

A

TFIIH

TFIIH is known for its role in phosphorylation which is crucial for transitioning into the elongation phase of transcription.

217
Q

What is one result of the phosphorylation of RNA polymerase II?

A

Release of many transcription factors

Phosphorylation allows RNA polymerase II to transition into the elongation phase by releasing transcription factors that are no longer needed.

218
Q

What enzyme does RNA polymerase II bind after phosphorylation?

A

Capping enzyme (guanylyl transferase)

The capping enzyme is involved in adding a protective cap to the 5’ end of the RNA transcript.

219
Q

What type of factors does RNA polymerase II bind to after phosphorylation?

A

Splicing factors

Splicing factors are essential for the RNA splicing process, which removes introns from the pre-mRNA.

220
Q

What apparatus does RNA polymerase II bind to after being phosphorylated?

A

Cleavage/polyadenylation apparatus

This apparatus is crucial for the processing of pre-mRNA by cleaving and adding a poly-A tail.

221
Q

What is the composition of the Mediator Complex?

A

At least 20 subunits

The Mediator Complex plays a crucial role in transcription regulation.

222
Q

Is the Mediator Complex required for basal transcription?

A

Not required

Basal transcription can occur without the Mediator Complex.

223
Q

What types of signals does the Mediator Complex transduce?

A

Positive and negative signals

These signals come from gene-specific activators and repressors.

224
Q

Can the Mediator Complex bind to DNA?

A

Cannot bind DNA

This characteristic differentiates it from some other transcription factors.

225
Q

What activity does the Mediator Complex regulate?

A

TFIIH CTD kinase activity

This regulation is important for the phosphorylation process during transcription initiation.

226
Q

What is the role of the promoter in transcription?

A

Sufficient for transcription at basal levels

The promoter is necessary for initiating transcription.

227
Q

How does the promoter act in relation to transcription?

A

In an orientation and position dependent manner

This means the promoter’s effectiveness can vary based on its location and orientation.

228
Q

What does the promoter bind to?

A

Components of transcription initiation complex

This binding is essential for the initiation of transcription.

229
Q

What factors may influence the promoter’s activity?

A

Factors bound at other DNA sites

These factors can enhance or repress the transcription process.

230
Q

What is one function of enhancers in gene expression?

A

Upregulate and downregulate transcription

Enhancers respond to extracellular activities and environmental signals.

231
Q

Fill in the blank: The Mediator Complex is required for _______.

A

[transcription regulation]

While not required for basal transcription, it is essential for regulated transcription.

232
Q

True or False: The Mediator Complex is essential for all types of transcription.

A

False

It is not necessary for basal transcription but is crucial for regulated transcription.

233
Q

What may the Mediator Complex bind to in relation to tissue-specific gene expression?

A

Some factors required for tissue-specific gene expression

This allows certain genes to be active only in specific tissues.

234
Q

What are enhancers in molecular biology?

A

Sequences not absolutely required for transcription that modulate the rate of transcription from a promoter.

235
Q

How do enhancers affect transcription levels?

A

They can increase or decrease transcription levels.

236
Q

What is the role of the glucocorticoid response element?

A

Involves higher levels of transcription.

237
Q

What is the effect of including additional bases around a promoter?

A

It results in more transcription.

238
Q

What is the S1 nuclease protection assay used for?

A

To study the components of typical transcriptional regulation.

239
Q

What is the relationship between transcription factors and transcription levels?

A

Transcription factors influence transcription through mechanisms like phosphorylation.

240
Q

What happens to histones during transcriptional activation?

A

Histones are acetylated.

241
Q

Fill in the blank: Enhancers are located in the _______ regions of genes.

A

flanking

242
Q

True or False: Transcription factors typically have multiple domains, including phosphorylation sites.

A

True

243
Q

What is the expected structure of transcription factors?

A

Dimerized with various domains including phosphorylation sites and dimerization groups.

244
Q

What is the significance of mediator in transcription?

A

It influences transcription factors like TFIIH and promotes phosphorylation.

245
Q

What are the components of typical enhancer binding proteins?

A

• DNA binding domain
• Dimerisation domain
• Transcriptional activation domain
• Ligand binding domain
• Phosphorylation / modification site

These components are crucial for the function of transcription factors in gene regulation.

246
Q

What is the function of the DNA binding domain in transcription factors?

A

It allows the transcription factor to bind to specific DNA sequences.

The DNA binding domain is essential for recognizing and attaching to the promoter or enhancer regions of genes.

247
Q

What is the role of the dimerisation domain in transcription factors?

A

Facilitates protein-protein interactions necessary for dimer formation.

Dimerization is important for the function of many transcription factors, allowing them to work together.

248
Q

What is the purpose of the transcriptional activation domain?

A

It mediates interactions with other proteins to activate transcription.

This domain enhances the transcriptional activity of RNA polymerase and other co-activators.

249
Q

What does the ligand binding domain do in transcription factors?

A

Binds to small molecules or ligands that can influence transcriptional activity.

The binding of ligands can lead to conformational changes that activate or repress transcription.

250
Q

What is the significance of the phosphorylation/modification site?

A

It allows for post-translational modifications that regulate transcription factor activity.

Phosphorylation can alter the stability, localization, or activity of transcription factors.

251
Q

What is the role of TAFs in transcription?

A

They are part of the transcription pre-initiation complex that assists RNA polymerase binding.

TAFs are important for recognizing promoter elements and facilitating the assembly of the transcription machinery.

252
Q

Which transcription factor is represented by TBP?

A

TATA-binding protein

TBP is a component of the transcription factor IID complex and is essential for the initiation of transcription.

253
Q

What does TFIIH do in the transcription process?

A

It is involved in the unwinding of DNA and phosphorylation of the RNA polymerase II C-terminal domain.

TFIIH has helicase activity, which is crucial for opening the DNA double helix during transcription initiation.

254
Q

What is the role of Mediator in transcription?

A

It serves as a bridge between transcription factors and RNA polymerase II.

Mediator helps integrate signals from various transcription factors to regulate gene expression.

255
Q

Fill in the blank: The DNA must be in an _______ format for transcription to occur.

A

open uncondensed

Open chromatin allows transcription factors and RNA polymerase access to the DNA.

256
Q

True or False: Transcription factors can influence transcription from locations that are 50,000 base pairs away.

A

True

This demonstrates the ability of enhancers to regulate transcription over long distances.

257
Q

What is the function of the polymerase along with the CDL tail?

A

It is recruited by TFIIH during transcription initiation.

The C-terminal domain (CDL) of RNA polymerase II is critical for its interaction with transcription factors.

258
Q

What are tissue-specific genes?

A

Genes that are expressed in only a single cell type

Examples include Growth Hormone in somatotroph cells and Prolactin in mammotroph cells of the pituitary gland.

259
Q

Which cell types express Growth Hormone?

A

Somatotroph cells in the anterior pituitary

Growth Hormone is present in every cell but is only active in somatotroph cells.

260
Q

Which cell types express Prolactin?

A

Mammotroph cells in the anterior pituitary

Prolactin is another example of a tissue-specific gene.

261
Q

How is gene transcription activated in specific cell types?

A

Factors only present in these cell types are required to activate the transcription of these genes

These factors often bind to specific DNA sequences in the gene’s promoter.

262
Q

True or False: All genes are expressed in every cell type of the body.

A

False

Many genes are only active in specific cell types, such as somatotrophs and mammotrophs.

263
Q

What is the role of DNA sequences in gene activation?

A

They are bound by proteins that are only found in specific cell types

This binding is crucial for the activation of tissue-specific genes.

264
Q

Fill in the blank: The likely way tissue-specific gene expression occurs is through a _______ in the promoter of these genes being bound by specific proteins.

A

DNA sequence

This process is essential for differentiating pluripotent stem cells into specific cell types.

265
Q

What is the significance of pluripotent stem cells in differentiation?

A

They can develop into various cell types, such as neurons or liver cells

Differentiation is triggered by specific signals during development.

266
Q

What is the function of activators in transcription?

A

They influence the transcription process to occur

Activators mediate the binding of RNA polymerase and other transcription factors.

267
Q

What are co-activators?

A

Proteins that assist activators in enhancing gene transcription

They do not bind to DNA directly but interact with other transcription factors.

268
Q

What are co-repressors?

A

Proteins that inhibit gene transcription

They bind to transcription factors and prevent the transcription machinery from assembling.

269
Q

What role do elongation factors play in transcription?

A

They assist RNA polymerase during the elongation phase of transcription

Elongation factors help in the process of synthesizing RNA from the DNA template.

270
Q

What are splicing factors?

A

Proteins involved in the splicing of pre-mRNA

They play a crucial role in the modification of RNA after transcription.

271
Q

What is the function of processing factors in RNA transcription?

A

They modify RNA transcripts after they are synthesized

This includes capping, polyadenylation, and splicing.

272
Q

What is RNA polymerase II responsible for?

A

Transcribing mRNA from DNA

It is essential for the synthesis of protein-coding genes.

273
Q

What is the function of the 5’ flanking region in the growth hormone gene?

A

It contains transcription factor binding sites for gene regulation.

This region is critical for the initiation of transcription by recruiting necessary proteins.

274
Q

Which transcription factor is specifically mentioned as binding to the GH promoter?

A

Pit-1

Pit-1 plays a crucial role in the expression of growth hormone.

275
Q

What is the consensus sequence for the binding elements in the growth hormone gene?

A

AAATATNCAT

This sequence is important for the binding of transcription factors to the promoter region.

276
Q

What are GRE-like elements associated with?

A

Glucocorticoid receptor binding

These elements are involved in the regulation of genes by glucocorticoids.

277
Q

Fill in the blank: The transcription factor _______ is essential for growth hormone gene expression.

A

Pit-1

278
Q

True or False: The growth hormone promoter is only influenced by proteins from the pituitary gland.

A

False

While pituitary extracts are mentioned, other factors may also influence the promoter.

279
Q

What is the significance of the TATAA sequence in gene promoters?

A

It serves as a core promoter element for transcription initiation.

The TATAA box is a common feature in many eukaryotic promoters.

280
Q

What does the term ‘unbound’ refer to in the context of transcription factors?

A

Transcription factors that are not currently attached to DNA sequences.

Unbound transcription factors can become active when conditions change.

281
Q

What role do consensus sequences play in gene regulation?

A

They provide specific binding sites for transcription factors.

This specificity is crucial for the precise regulation of gene expression.

282
Q

How does Pit-1 interact with the growth hormone promoter?

A

By binding to specific elements in the promoter region.

This interaction is vital for activating the transcription of the growth hormone gene.

283
Q

What is the role of transcription in molecular biology?

A

Transcription is the process of copying a segment of DNA into RNA.

284
Q

What type of cell is specifically mentioned in the context of transcription?

A

Pituitary cell.

285
Q

What is the result of transfecting a liver cell in this context?

A

No transcription occurs.

286
Q

What is involved in the process of transcription factor purification?

A

Isolating specific proteins that regulate transcription.

287
Q

What are the steps involved in gene cloning mentioned?

A

Sequence protein and clone gene.

288
Q

Fill in the blank: Transcription occurs in _______ cells.

A

[pituitary]

289
Q

True or False: Transcription occurs in liver cells after transfection.

A

False.

290
Q

What process is used to introduce foreign DNA into a cell?

A

Transfection.

291
Q

What is the purpose of fraction collection in molecular biology?

A

To isolate and analyze specific components from a mixture.

292
Q

What is the role of miRNA in translation inhibition?

A

miRNA inhibits translation of target RNA

miRNA can bind imperfectly to multiple mRNAs.

293
Q

What is the function of siRNA?

A

siRNA cleaves target RNA

siRNA is involved in the RNA interference pathway.

294
Q

What does the acronym PIT1 stand for?

A

PIT1 is a transcription factor from pituitary cells

It regulates gene expression in various tissues.

295
Q

True or False: A single miRNA can act on multiple mRNAs.

A

True

This is due to the imperfect binding of miRNA.

296
Q

Fill in the blank: miRNA can act on multiple _______.

A

mRNAs

This is a result of its imperfect binding ability.

297
Q

What happens to the expression of miRNA in cancers?

A

Cancers have dysregulation of miRNA

This can lead to altered gene expression and cancer progression.

298
Q

What is the significance of imperfect binding in miRNA function?

A

Imperfect binding allows a single miRNA to regulate multiple target RNAs

This increases the regulatory potential of miRNAs.

299
Q

Where does the PIT1 transcription factor originate?

A

PIT1 originates from pituitary gland cells

It is crucial for the development of certain hormones.

300
Q

What is the process of transfecting a gene of interest into a liver cell?

A

Transfection introduces foreign DNA into cells

This can be used to study gene function in non-native cells.

301
Q

What is the primary difference between miRNA and siRNA?

A

miRNA inhibits translation, while siRNA cleaves RNA

Both are involved in gene regulation but act through different mechanisms.

302
Q

What is the target of miR-196a in humans?

A

HOXB8 mRNA

miR-196a regulates HOXB8, which is involved in developmental processes.

303
Q

What is the target of miR-166 in plants?

A

PHAVOLUTA mRNA

miR-166 is important for plant development and leaf patterning.

304
Q

What is the overall impact of miRNA on gene expression?

A

miRNA can downregulate gene expression

This regulation can impact various cellular processes and pathways.

305
Q

What are miRNAs?

A

Short non-coding RNA molecules that regulate gene expression

miRNAs are crucial in post-transcriptional regulation.

306
Q

Approximately how many miRNA genes are believed to exist in the human genome?

A

~1000 genes

These miRNAs are involved in regulating a significant portion of gene expression.

307
Q

What proportion of human genes are regulated by miRNAs?

A

~1/3 of all human genes

This indicates the extensive role of miRNAs in gene regulation.

308
Q

How is miRNA made?

A

Through a process involving self-complementarity and the dicer complex

This process results in a double-stranded hairpin structure that is processed further.

309
Q

What is the role of the dicer complex in miRNA processing?

A

Removes the hairpin structure from the precursor miRNA

Dicer is essential for producing functional miRNA.

310
Q

What complex binds to the processed miRNA after dicer removes the hairpin?

A

RISC complex

RISC stands for RNA-induced silencing complex, which plays a crucial role in gene regulation.

311
Q

What is the major protein involved in the RISC complex?

A

Argonaute protein

Argonaute is integral to the functionality of RISC in gene silencing.

312
Q

What happens to the miRNA strand after it is processed by dicer?

A

Left with a single strand

This single strand is then ready to interact with target mRNAs.

313
Q

What are the two possible outcomes when a single strand of miRNA binds to a complementary strand?

A
  1. Perfectly bound = degradation of strand
  2. Imperfectly bound = failure to translate

These outcomes determine the effectiveness of gene silencing.

314
Q

Fill in the blank: The process of miRNA binding can lead to either degradation of the target strand or _______.

A

failure to translate

This highlights the role of miRNAs in regulating gene expression.

4.1 Molecular Biology (14 decks)

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