Lecture 24. Transcription Flashcards
What is transcription ?
How mRNA is made from a DNA template
What is transcriptomics?
Allows us to analyze gene sequences and expression levels of all genes in an organism, often simultaneously
What does RNAP II make in eukaryotes?
Makes mRNA
What does RNAP I make in eukaryotes ?
ribosomal RNA
What does RNAP III make in eukaryotes ?
Transfer RNA
What are the three steps in RNA synthesis ?
- Initiation of transcription
- Elongation
- Termination
What is a promoter ?
DNA sequences that guide RNAP to the beginning of a gene to start transcription at the initiation site
What is the function of a promoter ?
Tell RNA polymerase where and when to initiate transcription
What is transcription in eukaryotes regulated by ?
Transcription factors
What are the two types of transcription factor ?
- Repressors
2. Activators
What are 4 major differences between eukaryotic and prokaryotic transcription ?
- Nucleus surrounded by a double membrane
- DNA is wrapped around histones to give chromatin
- Promoters have many control sites for transcription factors
- RNAP has 12 protein subunits and can associate with many different regulatory proteins
What is involved in controlling transcription in eukaryotes ?
Opening and closing of chromatin
What is regulated by transcription factors and chromatin state ?
Access to core promoter
What is a proximal core promoter ?
General transcription factor complex substitute for sigma factor
Where is the TATA box analogous to ?
Pribnow box
What are distal control elements ?
Enhancer and silencer elements controlled by transcriptional activators/repressors influence general transcription factors an Pol II recruitment
What happens during pre-initiation complex assembly ?
- Binding of TATA binding protein and transcription factor IID
Where does the pre-initiation complex assemble around ?
RNA polymerase
Where does the TATA binding protein bind to ?
The TATA box in the genes promoter region
What does the general transcription factor form ?
A pre-initiation complex that includes RNAP
What are general transcription factors a complex version of ?
A sigma factor
What is RNA polymerase activated by ?
The phosphorylation of its C-terminal tail and then begins elongation/transcription
What does RNA polymerase II begin ?
Elongation
What does mRNA precursors contain ?
Exons and introns
Where are introns spliced ?
The nucleus
What is added to mRNA ?
- A 5’ CAP
2. 3’ PolyA tail
How is the mature mRNA transported ?
Transported from the nucleus to the cytoplasm via the nuclear pores
What are introns ?
Intervening regions
What are exons ?
Coding regions
What is splicing ?
Removal of introns
What are some things in mRNA in eukaryotes ?
- Exons
- Introns
- Capping
- Splicing
- Polyadenylation
What does alternative splicing yield ?
Different mRNA’s and protein isoforms
What is base pairing ?
Two highly negatively charged polymers held together
What is DNA hybridisation ?
A velcro type reaction, many low energy linkages multiply their effects to hold DNA together
What forms of DNA is unstable ?
Short DNA duplexes
What can be used to detect specific mRNAs ?
Northern blotting
What does electroblotting do ?
Drives the RNA out of the gel and onto the membrane
What is thermal stability of duplex directly related to ?
Perfection of base pair matching
What is the zippering effect ?
Longer duplexes hybridise faster and are more stable than short ones
What is hybridisation of probes ?
The basis of blotting techniques for detetion
What is the hybridisation of primer sequences ?
The basis for targeted DNA replication including PCR
What could a hybridised label of DNA of the gene of interest be labelled with ?
- Radioactive 32p
2. Biotin labelled nucleotides
What is reverse transcriptase ?
Makes DNA from an RNA template
What are microarrays used for ?
To measure changes in gene expression across the whole genome
How do you measure the abundance of mRNAs ?
Via amplification of cDNA
What can reverse transcription PCR be used for ?
Quantifies mRNA indirectly via amplification of cDNA copies
What are potential drug targets ?
Genes that are upregulated in cancer cells
What does each cells cDNA carry ?
A unique sequence tag
What can cell types be identified by ?
Identical transcriptomes
