Transcription and RNA Processing Flashcards
Summarize transcriptional activation in eukaryotes
TFs bind to the upstream DNA promoter activating sequences (UAS) and enhancer sequences, then attract HATs.
HMG proteins help it bend the chromatin structure so that TFs can interact with the Mediator, thus bridging between the transcriptional activators and the preinitiation complex (PIC)
TBP in transcriptional activation
Only binds one orientation on the TATA box of the promoter, so it orients the entire PIC ; specifies the correct strand for transcription ; and defines the transcriptional start site (+1)
TFIIE in transcription activation
ATPase and helicase activity (unwinding powered by ATP)
Also recruits TFIIH
TFIIH in transcriptional activation
- Helicase - unwinds DNA at promoter
- Kinase - phosphorylates Pol II CTD to activate PIC
- Recruits NER proteins
- Initiate PIC assembly in TATA-less promoters (initiator elements/DPE)
How is RNA polymerase activated over the transcription start site?
TATA binding protein (TBP) binds DNA in an orientation that assembles the PIC over the start site.
Specifically uses the template strand 3’ to 5’, so that transcription occurs 5’ to 3’.
Template/nontemplate, coding/noncoding, sense/antisense
Template = noncoding = antisense is used to synthesize the RNA
Nontemplate= coding = sense is identical to the RNA (substituting U for T)
What modifications occur to pre-mRNA at the 5’ end?
The 5’ triphosphate end of mRNA is methylated by cap synthesizing complex on the RNA Poly II CTD to form a 5’cap
The methyls groups are donated by SAM, whose regeneration requires folate and vitamin B12.
What’s the purpose of the 5’cap?
Stabilizes the mRNA
Helps promote translation
What modifications occur to premRNA at the 3’ end?
Addition of the poly A tail, as dictated by the poly A signal sequence upstream of the transcriptional termination
What’s the purpose of the 3’ poly A tail?
Stability
Facilitates nuclear export of the mRNa
Augments translation in the cytosol
What is the role of RNA Pol II CTD in pre-mRNA processing?
5’ capping is catalyzed by complexes associated with RNA pol II CTD, so that it occurs simultaneously with transcription.
Similarly, the endonuclease that cleaves the leftover transcript past the poly A tail is associated with the RNA pol II CTD
Splice donor site
at the start of the intron
splice acceptor site
at the end of the intron
What is the role of spliceosomes and snRNPs in RNA splicing?
The spliceosome compelx contains snRNPs that create a lariat structure that removes the intron, and joins the exons.
How do poisonous mushrooms affect transcription?
a-amanitin binds to RNA poly II’s back side and restricts its flexibility such that it can’t function –> significant reduction in RNA poly II transcripts and total protein
Liver damage, kidney failure, coma, death
How does HIV ensure full length RNA transcripts?
- Viral-encoded Tat protein acts as an anti-termination factor so that RNA Poly II keeps transcribing a full length viral transcript, which forms a 5’ hairpin loop.
- This loop binds viral Tat & host cyclin T and CDK9 proteins.
- Cyclin T activates CDK9 to hyperphosphorylate the CTD of RNA Poly II so prevent premature termination of the HIV transcript.
What is B thalassemia? How do you recognize it?
Mutation at the B-globin locus causes less B-globin and more y-globin to be transcribed and synthesized.
Decrease in hemoglobin A (a2B2)and increase in hemoglobin A2 (a2y2)
What’s the difference between B-thalassemia major (Cooley’s anemia), B thalassemia intermedia, and B+-thalassemia minor ?
Major : complete absence of B-globin expression; 2 alleles of Bo (Bo/Bo)
Intermedia: (B+/B+) or (Bo/B+)
Minor: reduced B-globin expression; least severe; (B+/B) or (Bo/B)
Bo = b globin levels are essentially zero
B+ = b lglobin levels are decreased
B = normal
How is RNA transcription and splicing involved in B-thalassemia?
B-thalassemia can be caused by
- Point mutations in UAS –> decreased transcription
- Mutations in splice donor or splice acceptor sites –> aberrant splicing
Systemic lupus Erythamatosis
Autoimmune disease where the body produces antibodies against its own components, including snRNPs, and triggers an inflammatory cascade –> multiorgan dysfunction
Spliceosome is made up of
snRNPs: complexes of snRNAs (U1,U2,U4,U5,U6) + proteins
What cuases myotonic dystrophy?
Expansion of CTG triplet repeats in the 3’UTR of the MDPK gene, which are transcribed into CUG repeats in mRNA, prevents nuclear export of the DMPK mRNA
The more repeats, the more severe
How are improper nuclear export of mRNA and splicing involved in myotonic dystrophy?
- As CUG repeats in the 3’UTR of the DMPK gene increase and can’t be exported, they build up in the nucleus and attract muscleblind.
- Since muscleblind is now bound to DMPK mRNA, it isn’t binding and acting as an inhibitor to the CUG-BP mRNA
- –> Increased expression of CUG-BP protein , which is involved in embryonic splicing
- –> increased aberrant splice forms of cardiac torponin T, insulin receptor, and Cl- channel –> abnormal thenar reflex, insulin resistance, myotonia
Where is the stop codon?
in the 3’-UTR. UAA
When does hnRNA get processed (5’cap, 3’polyA tail, splicing) into mRNA?
Simultaneous with transcription