L2: Gene Expression Basic Processes Flashcards
biological relevance of gene expression and regulation
- underlies many fundamental processes
- development
- cellular function
- organismal diversity
- human variation
- personalized medicine
- genetic disease
- cancer
therapeutic relevance of gene expression and regulation
- many therapies and biomedical technologies require understanding and manipulating of gene expression
- gene therapy
- recombinant DNA research
- production of biological therapeutics
- RNAi therapies
- CRISPR/Cas9
- Systems biology
- IPSCs
genetic differences in drug responsiveness
- lie in non-coding regions
- may affect expression of nearby genes
DNA -> RNA
- mRNA transcribed from template strand with complementary sequence
- occurs 5’ - 3’ in opposite orientation to template strand
where does RNA polymerase bind
- binds to the promoter
- elongation with RNA pol moving down DNA adding nucleotides to the 3’ end of the transcript
prokaryotic (bacterial) gene expression (and how different from eukaryotic)
- transcription and translation occur in same cellular compartment because there is no nucleus
- processes occur simultaneously
- no RNA splicing
- mRNAs are commonly polycistronic - multiple coding sequences to make different proteins
- multiple proteins from same transcript
- all genes transcribed by single RNA polymerase
- bacterial-induced disease
eukaryotic gene expression (and how different from bacterial)
- multiple compartments
- genes are monocistronic
- genes have introns and exons
- RNA splicing to eliminate introns
- different classes of genes (tRNA, rRNA, mRNA) transcribed by distinct RNA polymerases
- DNA packaged into chromatin
where eukaryotic transcription and RNA processing occurs
- nucleus
where eukaryotic translation occurs
- cytoplasm
exons
- coding and untranslated regions
RNA Pol I transcribes
- rRNA genes
- 28S
- 18 S
- 5.8S
RNA Pol II transcribes
- mRNA genes
- also microRNAs (regulation)
- snRNAs (RNA splicing)
RNA Pol III transcribes
- tRNA, 5S rRNA, additional small RNAs
- smaller genes
Are all three polymerases found in eukaryotes?
- yes found in all eukaryotes
Each RNA polymerase recognizes a different promoter that differs n sequence
what is the 4th RNA polymerase?
- mitochondria
- have their own
RNA polymerase II promoters
- where RNA polymerase and transcription starts
core promoters
- multiple types and elements
important class of core promoter
- TATA box - TATAA sequence
- 25 bp upstream of promoter
- binds TFIID
- initiator element (Inr) +1
TFIID
- TATA binding protein
- TBP + TAFs
enhancers
- bind TFs
- promote spatial, temporal, and quantitative control of transcription
upstream promoter-proximal elements
- GC rich regions - binds SP1 TF
- CAAT box - binds C/EBP
- regulate RNA levels
- bind transcription factors
- close to promoter (within 100 bp)
- elevate the levels of transcription
focused promoters
- focused at one site
- defined location
- contain TATA box and Inr elements
- regulated genes
- 20% of human promoters
dispersed promoters
- multiple start sites
- lie within CpG islands instead of TATA boxes
- housekeeping genes
- 80% of human promoters
- GC rich regions
housekeeping genes
- things that are pretty much on all the time.
- regulation of levels doesn’t change very much
regulated genes
- gene that gets turned on at specific times
RNA polymerase II composed of
- core enzyme composed of 12 subunits
General (Basal) transcription factors
- work with RNA polymerase to start eukaryotic transcription
- TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH
TATA binding factor
- TBF
- TBP-Associated Proteins (TAFs) - 13 proteins
TATA binding protein (TBP)
- part of TFIID
- binds TATA box
role of TFIIA and TFIIB
- stabilize TBP binding
- TFIIB positions and recruits RNA polymerase II over promoter
TFIIF role
- assists polymerase II binding
- followed by TFIIE and TFIIH
TFIIH role
- helicase
- unwinds DNA to open up helix and allow transcription to occur
- uses energy of ATP hydrolysis
TFIIH kinase role
- phosphorylates the C-terminal domain of polymerase II along with TFIIE
- RNA polymerase will not transcribe genes until phosphorylation occurs
- then transcription starts
which transcription factors are not released once transcription begins?
- TBP (remains at promoter to start another round of transcription)
- TFIIF (remains with Pol II)
First step of transcription initiation
- TFIID binds to TATA box
- because it contains TBP
- induces bending of DNA
second step of transcription initiation
- binding of TFIID is followed by binding of TFIIA and TFIIB
- stabilize TBP binding
- TFIIB positions and recruits RNA polymerase II over promoter at CRE and recruits TFIID
third step of transcription initiation
- RNA pol II recruited along with TFIIF
fourth step of transcription initiation
- TFIIE and TFIIF are recruited to form transcription initiation complex
what promoter does Pol I use?
- rlnr (ribosomal initiator) plus UPE/UCE sequences
- upstream promoter element
what promoter does Pol II use?
- TATA plus Inr (initiator)
- Inr plus DPE
- downstream promoter element
what promoter does Pol III use?
- internal sites (A and C for rRNA)
- A and B for tRNA
- has sequence elements that interact with pol III but are downstream of the genes
5S rRNA genes
- TFIIA and TFIIC recognize the promoter
tRNA gene
- TFIIC recognizes the promoter
other gene promoters
- use upstream TATA and PSE
- TATA recognized by TBP
related TBP-like proteins recognized by
- Pol I and Pol III
alpha amanitin toxin
- poisonous mushroom
- binds to RNA pol II and inhibits transcription
- causes severe kidney damage and can lead to death
prokaryotic transcription
- only one RNA polymerase for mRNA, tRNA, and rRNA genes
what does rifampin do?
- binds to the beta subunit of RNA pol and prevents initiation
- does not bind to eukaryotic RNA polymerases which is why we can use it on humans
prokaryotic promoters
- have two short conserved sequences (-35, -10) that are recognized by RNA polymerase
- looks like TATA box
prokaryotic RNA polymerase structure
- 5 subunits
- 2 alpha
- beta
- beta prime
- omega
sigma factors
- can vary
- allow holoenzyme (core + sigma) to recognize promoter
- allow it to target different promoters
leader
- 5’ untranslated region
- part of mRNA
trailer
- 3’ untranslated region
- has fairly extensive poly(A) tail that gets added on after the gene is transcribed
where do the untranslated regions come from
- exons that get spliced together
where does processing and modification of hnRNA occur?
- in the nucleus
hnRNPs
- interact with hnRNA to facilitate protein interactions, transport, and prevent degradation