Transcription Flashcards
What is the central dogma?
DNA codes for RNA. RNA codes for protein.
Flow of genetic information
How much RNA does Pol I make and which RNA?
90%
rRNA
How much RNA does Pol II make and which RNA?
10%
mRNA
How much RNA does Pol III make and which RNA?
1%
tRNA and other
How are Pol I and III regulated?
Depending on whether the cell is actively cycling or not
What are the general properties of RNA pol?
DNA directed RNA synthesis
RNA pol doesn’t need a primer
Transcription begins at specific sites
RNA produced 5’ to 3’
Properties of Pol I
rRNA transcription
Occurs in the nucleolus
rRNA genes occur in repeats - copies on several chromosomes
Properties of Pol II
mRNA transcription
Unwind DNA, RNA synthesis
Proof-reading
What can’t Pol II do?
Recognise promoter Initiate / terminate transcription Elongation Add 5' cap or polyA tail Splice out introns
Properties of Pol III
tRNA and 5s RNA transcription
Promoter is downstream - so forms part of the transcript
Nucleosome properties
147bp of DNA wraps around histone octamer
What is the histone octamer made up of?
(H2A, H2B, H3 and H4) x 2
Euchromatin properties
Open
Gene rich
Active gene
Unique sequences
Heterochromatin properties
Closed
Gene poor
Centromeres, telomeres
Repetitive sequences i.e. transposons
What is a transcription factory?
Nuclear substrates anchors multiple Pol II enzymes. Active genes are then recruited to the factory, rather than Pol II moving to each gene
How are chromosomes placed in the nucleus?
They each occupy a territory rather than being all mixed together
How does mRNA transcription begin?
With RNA Pol II binding to a promoter
What number is the transcription start site?
0
Why is the area around the transcriptional start site free of nucleosomes?
Maybe as RNA polymerase is there / so RNA polymerase can bind
What is the exception to the central dogma?
Reverse transcriptase used to convert mRNA to DNA
Why generate a cDNA library?
More stable than mRNA
Can sub-clone into plasmids and sequence
Where does RNA pol II bind?
To a promoter upstream of the gene
What’s different about cDNA compared to DNA?
All introns are spliced out so DNA sequence will not map the cDNA - there will be gaps
What is a reporter?
Something that can be measured e.g. an enzyme
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
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
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
Where is LCK expressed?
In the thymus - T-cells
What is the transcriptome?
The sum total of RNA present in the cell
How many protein-coding genes are in a given human cell and how many are active?
20,000 protein coding genes.
~30% are active
How can you look at mRNA?
Northern blot RT-PCR qRT-PCR Microarray RNA-seq
How can you look at protein?
Western blotting
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
How do you carry out northern blotting?
Radioactive DNA probe forms hybrid with RNA - gives band on gel
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
What are the cons of northern blotting?
Labour intensive
What is RT-PCR?
Reverse transcriptase PCR
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
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
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
What is epigenetics?
Study of modifications that alter phenotype without altering the genotype
Where does methylation occur in eukaryotes?
At CpG dinucleotides
What is the principle role of DNA methylation?
Switch off transcription long term
What are the two different kinds of DNA methyltransferases?
Dnmt1 - Replication dependent, maintenance
Dnmt 3a/3b - De novo
What is the exception to the gene silencing rule?
CpG islands - not methylated
How does DNA methylation lead to long term silencing?
Proteins bind to methylated cytosine. This interaction leads to gene silencing
What can be used to look at epigenetic modifications and proteins that bind to histones?
ChIP-seq
Chromatin immunoprecipitation - sequencing
In ChIP, what can you use antibodies to?
Histones, modified histones, transcription factors, histone modifying enzymes, chromatin remodelling proteins
What do transcription factors do?
Open or close chromatin to regulate transcription
What s SWI/SNF?
A remodeller
What is the structure of histones?
Globular domain
Long unstructured N-terminal tail
What does acetylating a lysine do?
Turn on transcription
neutralises the positive change so less association with DNA
How does an activator work?
Recruits co-activator with HAT activity
Adds acetyl groups to histone tails
Loosens tail
Makes space for polymerase
How does a repressor work?
Recruit co-repressors with HDAC activity
Removes acetyl group
Restores the charge
Tightens the chromosome
What binds to acetylated lysine?
Bromodomain
What binds to methylated lysine?
Chromodomain
PhD finger
How does H3K9 signal for euchromatin?
Acetylated - Bromodomain binds as part of P/CAF which is a HAT
How does H3K9 signal for heterochromatin?
Methylated - Chromodomain binds which recruits HP1 which attracts Dnmt3a
What can be used to look at histone-modifications?
ChIP-seq
What are the 4 steps of transcription?
- Recruitment of polymerase to promoter
- Initiation of transcription
- Clearance of polymerase from promoter
- Elongation of transcript
What are the general transcription factors?
TFIID TFIIB TFIIF TFIIE TFIIH
What does TFIID contain?
TBP (TATA binding protein) and 13 TAFs
What does TFIIB do?
Binds TBP, Pol II and promoter DNA
Helps fix transcription start site
What does TFIIF do?
Binds the coding strand to keep the ‘bubble’ open
What does TFIIE do?
Recruits TFIIH
What does TFIIH do?
Helicase activity unwinds DNA strands.
Kinase activity removes mediator
What are the core promoter elements sin addition to the TATA box?
Initiator sequence
Core promoter element
TFIIB recognition element
How are transcriptional programmes maintained?
Epigenetic modifications
What does the stability of cell state rely on?
Silencing of genes encoding regulators of other cell states
How do you reprogram the transcriptome?
Replace master transcription factor
What is big obstacle to reprogramming?
Epigenetic modifications - mammalians cells do not do demethylation
