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BS3010 Gene Expression UOL > Transcription > Flashcards

Transcription Flashcards

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
Q

How do you carry out a reporter assay?

A

Reporter contained on plasmid. Take plasmids and put into cell line of interest. The more transcription, the more reporter gene being expressed

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26
Q

What is saturation mutagenesis?

A

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

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27
Q

How do you carry out ChIP-seq?

A

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

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28
Q

Where is LCK expressed?

A

In the thymus - T-cells

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29
Q

What is the transcriptome?

A

The sum total of RNA present in the cell

30
Q

How many protein-coding genes are in a given human cell and how many are active?

A

20,000 protein coding genes.

~30% are active

31
Q

How can you look at mRNA?

A 
Northern blot
RT-PCR
qRT-PCR
Microarray
RNA-seq
32
Q

How can you look at protein?

A

Western blotting

33
Q

Why is mRNA used to monitor gene expression?

A

Can be probed with high specificity due to base pairing of nucleic acid
Many transcripts can be probed simultaneously from the same sample

34
Q

How do you carry out northern blotting?

A

Radioactive DNA probe forms hybrid with RNA - gives band on gel

35
Q

What are the pros of northern blotting?

A

Definitive experiment to test mRNA levels
Can measure size of transcript
Can detect if alternative splicing has taken place
Inexpensive

36
Q

What are the cons of northern blotting?

A

Labour intensive

37
Q

What is RT-PCR?

A

Reverse transcriptase PCR

38
Q

Method of qRT-PCR?

A

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
Q

What is the method of generating 1st generation microarrays?

A

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
Q

What is the method of generating RNA-seq?

A 
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
Q

What is epigenetics?

A

Study of modifications that alter phenotype without altering the genotype

42
Q

Where does methylation occur in eukaryotes?

A

At CpG dinucleotides

43
Q

What is the principle role of DNA methylation?

A

Switch off transcription long term

44
Q

What are the two different kinds of DNA methyltransferases?

A

Dnmt1 - Replication dependent, maintenance

Dnmt 3a/3b - De novo

45
Q

What is the exception to the gene silencing rule?

A

CpG islands - not methylated

46
Q

How does DNA methylation lead to long term silencing?

A

Proteins bind to methylated cytosine. This interaction leads to gene silencing

47
Q

What can be used to look at epigenetic modifications and proteins that bind to histones?

A

ChIP-seq

Chromatin immunoprecipitation - sequencing

48
Q

In ChIP, what can you use antibodies to?

A

Histones, modified histones, transcription factors, histone modifying enzymes, chromatin remodelling proteins

49
Q

What do transcription factors do?

A

Open or close chromatin to regulate transcription

50
Q

What s SWI/SNF?

A

A remodeller

51
Q

What is the structure of histones?

A

Globular domain

Long unstructured N-terminal tail

52
Q

What does acetylating a lysine do?

A

Turn on transcription

neutralises the positive change so less association with DNA

53
Q

How does an activator work?

A

Recruits co-activator with HAT activity
Adds acetyl groups to histone tails
Loosens tail
Makes space for polymerase

54
Q

How does a repressor work?

A

Recruit co-repressors with HDAC activity
Removes acetyl group
Restores the charge
Tightens the chromosome

55
Q

What binds to acetylated lysine?

A

Bromodomain

56
Q

What binds to methylated lysine?

A

Chromodomain

PhD finger

57
Q

How does H3K9 signal for euchromatin?

A

Acetylated - Bromodomain binds as part of P/CAF which is a HAT

58
Q

How does H3K9 signal for heterochromatin?

A

Methylated - Chromodomain binds which recruits HP1 which attracts Dnmt3a

59
Q

What can be used to look at histone-modifications?

A

ChIP-seq

60
Q

What are the 4 steps of transcription?

A
  1. Recruitment of polymerase to promoter
  2. Initiation of transcription
  3. Clearance of polymerase from promoter
  4. Elongation of transcript
61
Q

What are the general transcription factors?

A 
TFIID
TFIIB
TFIIF 
TFIIE
TFIIH
62
Q

What does TFIID contain?

A

TBP (TATA binding protein) and 13 TAFs

63
Q

What does TFIIB do?

A

Binds TBP, Pol II and promoter DNA

Helps fix transcription start site

64
Q

What does TFIIF do?

A

Binds the coding strand to keep the ‘bubble’ open

65
Q

What does TFIIE do?

A

Recruits TFIIH

66
Q

What does TFIIH do?

A

Helicase activity unwinds DNA strands.

Kinase activity removes mediator

67
Q

What are the core promoter elements sin addition to the TATA box?

A

Initiator sequence
Core promoter element
TFIIB recognition element

68
Q

How are transcriptional programmes maintained?

A

Epigenetic modifications

69
Q

What does the stability of cell state rely on?

A

Silencing of genes encoding regulators of other cell states

70
Q

How do you reprogram the transcriptome?

A

Replace master transcription factor

71
Q

What is big obstacle to reprogramming?

A

Epigenetic modifications - mammalians cells do not do demethylation

BS3010 Gene Expression UOL (3 decks)

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