Transcription Flashcards
Tell me about rRNA
- comprises 80% of cellular RNA
- transcribed by RNAPI
- are 5S, 5.8S, 18S, and 28S subunits of ribosome
Template-product relationship b.w. DNA and mRNA
RNA product looks like the coding strand (not template)
RNAP Prokaryotic Holoenzyme
- 2 Alphas, 2 Betas, 1 omega subunits + sigma factor
- several different sigma subunits bind different promoter sequences
Transcription Initiation in Prokaryotes
- primary pt of regulation in prokaryotic transc.
- sigma subunit binds -10 (Pribnow) and -35 regions
- DNA gyrase resolves + supercoiling
- TopoI resolves - supercoiling
- no primer needed
- when RNAP starts, sigma factor is left behind
Rho Dependent vs. Rho Independent Termination
- Rho protein binds specific RNA sequence as hexamer and contacts RNAP, causing dissociation
- Independent: RNA hairpins generated by palindromic sequences followed by U rich sequences cause RNAP to fall off.
Operator in Prokaryotic Transcription Regulation
- usually overlaps promoter
- negative regulatory element that binds repressor protein, which causes RNA to not bind promoter
Rifampicin
- binds beta subunit of RNAP and inhibits initiation
- treats tuberculosis
Actinomycin
- binds DNA template, phenoxazone ring intercalates b.w. Adjacent GC bps
- blocks elongation
- also interferes with eukaryotes, making it cancer treatment
Eukaryotic Promoter Elements
- 25 TATA box, the nucleation site
- promoter proximal elements and enhancers are more upstream
- PPEs are closer, binding sites for TFs of housekeeping genes. SP1 is a well categorized activator.
- enhancers can be upstream, downstream, or even within a gene
Tata Binding Protein
Part of TFIID
-binds TATA box, causes bend in DNA, allowing TFIIB to bind
TFIIB
Binds bent promoter DNA/TFIID
Allows RNAPII + TFIIF to assemble
TFIIE and TFIIH
Bind to DNAPII during initiation to help promoter melting
5’ Cap
- 7-methylguanosine covalently linked to 5’ end of mRNA via 5’–>5’ triphosphate linkage
- not encoded by DNA template
- added to mRNA shortly after initiation
- recognized by translation machinery to facilitate protein synthesis
Poly-A Tail
- happens during termination
- specified by DNA seuqnce
- endonucleolytic cleavage of nascent transcript
- addition of 100-250 residue poly-a tail
Steroid Hormone Receptors (structure and mechanisms of action)
- have hormone binding and DNA binding domains
- upon hormone binding, conformational change to allow for DNA binding and to create transcriptional activation domain that binds coactivator complex
2 cases:
Soluble in cytosol
Upon binding hormone, exposure of NLS
Enters nucleus, acts on DNA
Receptor is already in nucleus
Hormone binding either stimulates receptor-DNA binding or induces conformational change in receptor-DNA complex
BOTH REACT WITH COACTIVATOR COMPLEX TO STIMULATE RNA POL II
Steroid Response Elements
- enhancers for steroid responsive genes
- palindromic
Mechanisms of Transcriptional Activation
- some recruit or stabilize RNAPII/GTP to core promoter
- some clear a path by altering chromatin DNA template
Coactivators
2 Classes of Chromatin Modifiers
- catalyzes covalent modification of N-termini of histone tails
- HATs (N termini of H3 and H4) and HDACs
- some methlate, phosphorylate, ubiquitinate
- ATP dependent nucleosome remodeling complexes
- do not catalyze histone modification
MMTV
Mouse mammary tumor virus
DNA virus that causes breast epithelial cell carcinoma
Genome includes sequence identical to estrogen enhancer
Integration of MMTV into human genome leads to cancer
Integrates adjacent to wnt-1-proto-oncogene, placing the regulator of cell proliferation under control of estrogens
Tamoxifen
Antagonist of estrogen receptor
Fails to induce the conformational change in estrogen receptor that is required for subsequent coactivator binding
TFIIH
Plays important role in coupling transcription with DNA damage repair
Altered TFIIH leads to XP, TTD, and CS
Alpha-Amanitin
- binds tightly and specifically to RNAPII
- used to distinguish b.w. RNAPII from I and II
- inhibits elongation
When does splicing occur?
-either co or post-transcriptionally, but always before translation
Mechanism of pre-mRNA splicing
Factors That Contribute to Splicing Fidelity
- base sequence of intron begins with GU and ends with AG
- consensus sequences are found at 5’ and 3’ ends of introns
- branch sequence is found 20-50 nt away from 3’ end of intron
- catalyzed by spliceosome, assembled denovo from SNURPs on precursor RNA that is ATP dependent
- NO ATP is needed for actual transesterification
2 transesterification rxns
- cleavage of PDE bond between exon 1 and intron 1
- rxn uses 2’OH of branch pt A to form 2’-5’ PDE bond - 3’-OH of exon 1 cleaves PDE bond bw the intron and exon2
- exon 1 is now joined to exon 2 and intron is released as lariat
