RNA Structure And Synthesis Flashcards
Amount of RNA in cell vs DNA
40x more RNA than DNA in the cell
DNA vs RNA
DNA
- master copy
- carefully reproduced
- stable
- nucleus
RNA
- working copy
- so tangly synthesized, used and degraded
- cytoplasm
Which is larger: eukaryotic cell or bacterial cell?
Eukaryotic cell
Which is larger: mRNA or primary transcript?
Primary transcript
Similarities between DNA and RNA structures
- long unbranched polymers of nucleotides linked by 3’-5’ phosphodiester bonds
- sugar phosphate backbone
- 3 common bases: adenine, guanine, cytosine
Difference in DNA and RNA structures
- sugar in RNA is ribose
- RNA contains uracil instead of thymine
- RNA is shorter than DNA
- always single stranded
- RNA can form secondary loop and stem structures (i.e. tRNA)
Sort types of RNA from most abundant to least
rRNA –> tRNA –> mRNA
Basics of Transcription
Sequence dependent
Anti-parallel direction for DNA and RNA
- DNA read 3’ –> 5’
- RNA synthesized 5’ –> 3’
Asymmetrical: only one strand is transcribed
Does not require a primer
Coding (sense) strand
the DNA strand that goes 5’ –> 3’
The sequence that correlates with the mRNA
Template (non-coding) strand
DNA strand that goes 3’ –> 5’
Strand that is copied during RNA synthesis
Beta subunit on RNA polymerase
Contains the catalytic site- catalyzes 3’ –> 5’ phosphodiester bonds
Alpha subunit on bacterial RNA polymerase
Recognizes promoter regions, which is required for appropriate initiation of transcription
Does RNA polymerase transcribe the entire genome?
Nope
Subunits of the core enzyme
Alpha (2) (interacts with proteins)
Beta
Beta’ (binds DNA template)
Subunits of the holoenzyme
Same as core + sigma
Is transcription discontinuous or continuous?
It’s discontinuous –> different start/stop signals
Either strand can be a coding template
Prokaryotic Promoter Region
Upstream of the start site of transcription initiation (++1)
Written on the sense strand
Specifically recognized by sigma subunit
Pribnow sequence and -35 region : signals RNA polymerase to come and start transcription
What is important for efficient initiation of transcription?
The distance between pribnow box and the -35 region
Not every gene has the same Pribnow and -35 sequence
Transcription: Initiation
Sigma binds to specific promoter regions of DNA (-35 and -10)
These regions correspond with DNA sequence…if there was a different sequence in between, sigma would not bind
Steps in transcription initiation
1) RNA pol binds to DNA- RNA pol for enzyme is always bound to DNA in a non-specific manner
2) when sigma subunit binds to core enzyme at promoter site, a conformational change occurs and binding is stabilized (closed promoter complex)
3) After formation of the closed promoter complex, the 2 strands of the DNA template unwind to produce and unpaired region containing approximately 12 bases (open promoter complex)
4) 1st base in RNA chain is added complementary to the base at +1 of the template strand
5) After 8-10 bases are added, the sigma subunit leaves the complex and only the core enzyme remains
Role of beta subunit of RNA polymerase in initiation
Beta subunit has 2 nucleotide binding sites
1) for 5’ purine (therefore first base is a 5’ purine)
2) for subsequent NTPs
Also synthesized the phosphodiester 3’–5’ bond between NTPs
Transcription Elongation
RNA-DNA hybrid about 8 bases long
Transcription bubble always 12 bases
As the enzyme moves to the region in front, creates + super coils and the region behind it re-winds creating negative supercoils
Hairpin formation in transcription termination
Must be a GC rich palindrome (very stable)
Must be followed by UUUs
Rho-independent termination of transcription
Pol slows down at GC rich region
Hairpin forms
Weak A/U bonds
How does the inverted repeat of rho independent termination end?
Stops at T-A spots
What is Rho
Rho is a large hexameric protein-Helicase, ATPase
Works in the 5’ –> 3’ direction
Rho-dependent Termination of Transcription
Rho binds to the RNA transcript
Polymerase slows at a C-rich pre-termination sequence
Rho moves to the 3’ end of the RNA transcript, hydrolyzing ATP
Uses Helicase activity to unwind DNA-RNA hybrid–> to release RNA from complex
Dissociation of Rho and RNA polymerase from the RNA
Key differences between transcription in eukaryotic vs prokaryotic cells
Eukaryotes have:
3 polymerase so
No sigma subunit
Polymerase-protein interactions, not pol-DNA interactions govern transcription initiation
RNA polymerase II
Has no sigma factor to locate promoter region but interacts with specific proteins which bind core promoter regulatory elements
Transcribes the precursors of mRNAs (primary transcript)
Enables specific genes to be expressed in different developmental stages, different tissues, and in response to different environmental stimuli
RNA Pol LL Promoter/Enhancer Regions
More complicated than prokaryotes –> there aren’t any promoter sequences or organization like that
Enhancer- regulates efficiency of transcription
Constitutive- will be specific to the tissue its in
Basal- where it starts
TATA box- present but not in all genes (determines where transcription starts)
everything depends on tissue and what sort of gene is being transcribed
Basal Transcription Complex- Eukaryotes
TFII- transcription factors for Pol II
TBP- TATA binding protein
TAF- TATA associated factors
Protein-protein interactions are key to initiation
Formation of open promoter complex involves ATP hydrolysis
TFIIH= kinase which phosphorylates Pol II
Transcription initiation
Transcription elongation- basal apparatus
Elongation phase similar to that in prokaryotes
Dephosphorylation involved in termination
Inducible region
Specificity of Tx
Hormone receptor binding
Enhancer Region
Activator binds
CAAT Boc
Stabilized binding of pol 2
Controls of Tx (along with GC box)
What allows for efficient transcription?
The 3D structure of protein and polymerase II and the gene
Chromatin remodeling, histone acetylation/deacetylation also involved in efficiency of transcription initiation
Use of Tamoxifen
In order to identify potential gene targets, an estrogen receptor positive breast cancer cell line grown in culture is given tamoxifen
RNA is slanted from untreated cells and cells treated with different doses of tamoxifen and microarray analysis is performed to identify upregulated (red) and down regulated (green) genes
The upregulated genes are likely regulated by binding of the ER tamoxifen complex to an Inducible element (i.e.: tamoxifen)
Alpha- amanitin
Inhibits RNA Pol II»_space;> Pol III not Pol I
No mRNA = no protein synthesis = liver failure, death
