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BS3010 Gene Expression UOL > Transcription > Flashcards

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1
Q

What is the central dogma?

A

DNA codes for RNA. RNA codes for protein.

Flow of genetic information

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2
Q

How much RNA does Pol I make and which RNA?

A

90%

rRNA

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3
Q

How much RNA does Pol II make and which RNA?

A

10%

mRNA

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4
Q

How much RNA does Pol III make and which RNA?

A

1%

tRNA and other

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5
Q

How are Pol I and III regulated?

A

Depending on whether the cell is actively cycling or not

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6
Q

What are the general properties of RNA pol?

A

DNA directed RNA synthesis
RNA pol doesn’t need a primer
Transcription begins at specific sites
RNA produced 5’ to 3’

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7
Q

Properties of Pol I

A

rRNA transcription
Occurs in the nucleolus
rRNA genes occur in repeats - copies on several chromosomes

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8
Q

Properties of Pol II

A

mRNA transcription
Unwind DNA, RNA synthesis
Proof-reading

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9
Q

What can’t Pol II do?

A 
Recognise promoter
Initiate / terminate transcription
Elongation
Add 5' cap or polyA tail
Splice out introns
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10
Q

Properties of Pol III

A

tRNA and 5s RNA transcription

Promoter is downstream - so forms part of the transcript

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11
Q

Nucleosome properties

A

147bp of DNA wraps around histone octamer

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12
Q

What is the histone octamer made up of?

A

(H2A, H2B, H3 and H4) x 2

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13
Q

Euchromatin properties

A

Open
Gene rich
Active gene
Unique sequences

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14
Q

Heterochromatin properties

A

Closed
Gene poor
Centromeres, telomeres
Repetitive sequences i.e. transposons

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15
Q

What is a transcription factory?

A

Nuclear substrates anchors multiple Pol II enzymes. Active genes are then recruited to the factory, rather than Pol II moving to each gene

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16
Q

How are chromosomes placed in the nucleus?

A

They each occupy a territory rather than being all mixed together

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17
Q

How does mRNA transcription begin?

A

With RNA Pol II binding to a promoter

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18
Q

What number is the transcription start site?

A

0

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19
Q

Why is the area around the transcriptional start site free of nucleosomes?

A

Maybe as RNA polymerase is there / so RNA polymerase can bind

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20
Q

What is the exception to the central dogma?

A

Reverse transcriptase used to convert mRNA to DNA

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21
Q

Why generate a cDNA library?

A

More stable than mRNA

Can sub-clone into plasmids and sequence

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22
Q

Where does RNA pol II bind?

A

To a promoter upstream of the gene

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23
Q

What’s different about cDNA compared to DNA?

A

All introns are spliced out so DNA sequence will not map the cDNA - there will be gaps

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24
Q

What is a reporter?

A

Something that can be measured e.g. an enzyme

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25
How do you carry out a reporter assay?
Reporter contained on plasmid. Take plasmids and put into cell line of interest. The more transcription, the more reporter gene being expressed
26
What is saturation mutagenesis?
Changing every nucleotide in the sequence on at a time and then measuring if there are any changes by measuring activity of reporter gene. Can then see what binds to the sequence to induce changes
27
How do you carry out ChIP-seq?
Cross link cells chemically so protein will remain bound to DNA Fragmentation Add protein of interest Immunoprecipitation Digest protein Add adapters and then sequence. Map sequence against known genome
28
Where is LCK expressed?
In the thymus - T-cells
29
What is the transcriptome?
The sum total of RNA present in the cell
30
How many protein-coding genes are in a given human cell and how many are active?
20,000 protein coding genes. | ~30% are active
31
How can you look at mRNA?
``` Northern blot RT-PCR qRT-PCR Microarray RNA-seq ```
32
How can you look at protein?
Western blotting
33
Why is mRNA used to monitor gene expression?
Can be probed with high specificity due to base pairing of nucleic acid Many transcripts can be probed simultaneously from the same sample
34
How do you carry out northern blotting?
Radioactive DNA probe forms hybrid with RNA - gives band on gel
35
What are the pros of northern blotting?
Definitive experiment to test mRNA levels Can measure size of transcript Can detect if alternative splicing has taken place Inexpensive
36
What are the cons of northern blotting?
Labour intensive
37
What is RT-PCR?
Reverse transcriptase PCR
38
Method of qRT-PCR?
A short nucleotide probe with a Reporter-Quencher binds to cDNA The reporter is cleaved from the quencher by the nuclease activity of Taq polymerase PCR machine measures fluorescence intensity every cycle
39
What is the method of generating 1st generation microarrays?
DNA or RNA isolated from both samples, transformed and amplified into fluorescently labelled cDNA / cRNA Mix labelled samples and hybridise onto microarrays and scan
40
What is the method of generating RNA-seq?
``` Take mRNA, turn in cDNA Break into small fragments Ligate primers on to each end The more times that fragment is present, the more sequence reads you will get Map back to reference genome ```
41
What is epigenetics?
Study of modifications that alter phenotype without altering the genotype
42
Where does methylation occur in eukaryotes?
At CpG dinucleotides
43
What is the principle role of DNA methylation?
Switch off transcription long term
44
What are the two different kinds of DNA methyltransferases?
Dnmt1 - Replication dependent, maintenance | Dnmt 3a/3b - De novo
45
What is the exception to the gene silencing rule?
CpG islands - not methylated
46
How does DNA methylation lead to long term silencing?
Proteins bind to methylated cytosine. This interaction leads to gene silencing
47
What can be used to look at epigenetic modifications and proteins that bind to histones?
ChIP-seq | Chromatin immunoprecipitation - sequencing
48
In ChIP, what can you use antibodies to?
Histones, modified histones, transcription factors, histone modifying enzymes, chromatin remodelling proteins
49
What do transcription factors do?
Open or close chromatin to regulate transcription
50
What s SWI/SNF?
A remodeller
51
What is the structure of histones?
Globular domain | Long unstructured N-terminal tail
52
What does acetylating a lysine do?
Turn on transcription | neutralises the positive change so less association with DNA
53
How does an activator work?
Recruits co-activator with HAT activity Adds acetyl groups to histone tails Loosens tail Makes space for polymerase
54
How does a repressor work?
Recruit co-repressors with HDAC activity Removes acetyl group Restores the charge Tightens the chromosome
55
What binds to acetylated lysine?
Bromodomain
56
What binds to methylated lysine?
Chromodomain | PhD finger
57
How does H3K9 signal for euchromatin?
Acetylated - Bromodomain binds as part of P/CAF which is a HAT
58
How does H3K9 signal for heterochromatin?
Methylated - Chromodomain binds which recruits HP1 which attracts Dnmt3a
59
What can be used to look at histone-modifications?
ChIP-seq
60
What are the 4 steps of transcription?
1. Recruitment of polymerase to promoter 2. Initiation of transcription 3. Clearance of polymerase from promoter 4. Elongation of transcript
61
What are the general transcription factors?
``` TFIID TFIIB TFIIF TFIIE TFIIH ```
62
What does TFIID contain?
TBP (TATA binding protein) and 13 TAFs
63
What does TFIIB do?
Binds TBP, Pol II and promoter DNA | Helps fix transcription start site
64
What does TFIIF do?
Binds the coding strand to keep the 'bubble' open
65
What does TFIIE do?
Recruits TFIIH
66
What does TFIIH do?
Helicase activity unwinds DNA strands. | Kinase activity removes mediator
67
What are the core promoter elements sin addition to the TATA box?
Initiator sequence Core promoter element TFIIB recognition element
68
How are transcriptional programmes maintained?
Epigenetic modifications
69
What does the stability of cell state rely on?
Silencing of genes encoding regulators of other cell states
70
How do you reprogram the transcriptome?
Replace master transcription factor
71
What is big obstacle to reprogramming?
Epigenetic modifications - mammalians cells do not do demethylation

BS3010 Gene Expression UOL (3 decks)

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