RNA Synthesis & Splicing Flashcards
What are the 3 major ways in which most pre-mRNA’s are Processed?
Capping
Splicing
Clevage/polyadenylation
What enzymes are involved in capping?
Triphosphatase (removes terminal 5’ phosphate)
Guanylltransferase (adds GMP backwards: 5’-5’ linkage)
Guanine 7 methyltransferase (universal methyl transfer enzyme- adds methyl group from s-adenosylmethionine)
Describe the 2 steps of splicing (think lariat formation)
- Intron: 3’ A attacks 5’ GU (attack of 2’ –OH of branchpoint A)
- 3’ –OH of exon 1 attacks 5’ end of exon2
➢5’ splice site recognized by base pairing to U1snRNA
➢Branch point recognized by base pairing at U2 snRNA
What are the 2 enzymatic steps that add the 3’ polyA tail?
- clevage – endonucleic cutting (~3bp from DNA AATAAA) – exposes 3’ hydroxyls
- polyadenylation – exposed 3’ hydroxyl becomes addition site for unadenylated AAA’s (2-300)
What process is coupled with the 3’ end formation of the polyA tail?
Termination of transcription by RNA polI
What are the functions of the 5’ cap?
➢ Protects mRNA from ribonucleases, stabilizes against exonucleases
➢ Recognition site for binding proteins -> heterodimers (enhance export product)
➢ De-capped = degredation of mRNA
What is the conserved/ consensus 5’ sequence of introns?
(exon A G): G U A/G A G U
What is the conserved/ consensus 3’ sequence of introns?
U A C U A C
What is the conserved/ consensus sequence at the polyA tail?
A A U A A A
What are the 5 mechanisms of alternative splicing?
- Exon removed “skipping”
- Mutually exclusive exons
- Exon truncation/extension at 5’ end
- Eon trunication/extension at 3’ end
- Intron retained v. removed
What are 2 genetic disorders caused by splicing defects?
Marfan Syndrome (Abe Lincon)
- Dominant autosomal
- Mutation disrupts splicing of fibrillin gene transcripts (connective tissue)
- Big, strong, tall
- Weak aorta, protruding sternum
Spinal Muscular Atrophy (fatal before 2yo)
- SMN1 gene – 2 copies if 1 a dud- have phenotype
- Single amino acid difference at exon 7=> causes high frequency of skipping exon 7
- Shorter protein that doesn’t do its job
What codon starts transcription? What amino acid does it code for?
AT(U)G
Methionine (Met)
What are the 2 main functins of the polyA tail?
stabilization -> prevent degredation
enhance transcription
How can alternative polyA sites be used to make >1 protein from a single gene?
earlier or later = longer or shorter AA/ protein
Altered polyA site choice in cancer cells:
-Shortening of 3’UTR by alternative clevage/adenylation activates oncogenes in cancer cells
-Proliferating cells express mRNA’s with short 3’ UTR and fewer microRNA targets (microRNA is postranscriptional control of cell)
What 5 steps in the transcription cycle are common to prokaryotic and eukaryotic RNA polymerase?
- (Initiation) “Closed Complex” formed when polymerase binds to promoter sequence of double stranded DNA.
- (Initiation) “Open Complex” formed as polymerase melts apart double stranded DNA near transcription start site
- (Initiation) Polymerase catalyzes phosphodiester linkage(this is the precise moment of initiation) of two initial rNTPs (rNTP: ribonuclotide triphosphate – building block of RNA synthesis)
- (Elongation) RNA polymerase travels 3’-5’ on template (non-coding) strand [RNA IS SYNTHESIZED IN 5’ TO 3’ DIRECTION] -> travels down template strand -> melts DNA duplex, adds rNTPs and grow RNA
- Punctuated by starting and stopping
- Promoters escape shortly after elongation begins - (Termination) At transcription stop site CTD of polymerase is dephosphorylated and polymerase releases complete RNA, dissociates from DNA
Name the RNA polymerases and their main functions
RNA pol I
- rRNA (precursors of 18s, 5.8s & 28s ribosomal subunits)
- Busiest in growing cells
- 90% by weight of RNA is rRNA
RNA pol II
- Mainly mRNA
- Also snRNA, miRNA
- lncRNA: long non-coding RNA w regulatory function, inactivation of one X chromosome in each cell of males
- Reognizes thousands of sequences with large variation in sequence – usually have TATA box (consensus sequence)
RNA polIII
- tRNA
- 5s rRNA
- Well understood promoters (sometimes located within gene itself)
Define a promoter and name sequence elements characteristic of promoters in human genes .
Promoters direct transcription of adjacent segments of DNA
- DNA bp before RNA transcription -, bp after transcription +
- Consensus sequence-> similar or identical sequence with same meaning
- TATA box at -30 bp
- INR = initiation sequence where DNA is unwould +1: YYAN(A/T)YY
- N – any nucleotide
- Y - pyrimidine
- Promotors can be hundreds or thousands of bp away from transcription start site
How do α-amanitin and rifampicin block transcription?
α-amanitin
- From death cap mushroom
- Amanita phalloides is non-competitive inhibitor of RNA pol II -> Binds bridge helix and blocks RNA chain elongation=> prevents translocation
- “constipation” of transcribed RNA that cannot exit cell
Rifampicin
- Broad spectrum anti-biotic
- Binds bacterial RNA polymerase and blocks RNA exit channel
- Blocks elongation
- Used in microbacterial TB but resistance is an issue so regulated use as part of multi-pronged tx plan
Name 4 components of RNA pol II pre-initiation complex
- TFIIA – Stabilizes binding of TFIIB and TBP to promoter
- TFIIB – Binds to TBP, recruits TFIIF-pol II complex
- TFIIE –Recruits TFIIH, ATPase and Hlicase activities
- TFIIF – Bind pol II tightly, binds TFIIB, prevents pol II from binding non-specifically to DNA
- TFIIH – Unwinds DNA at promoter, phosphorylates pol II, recruits nucleotide-excision repair proteins
TBP/ TATA binding protein – specifically recognizes TATA box => recurits pre-initiation complex
What process is coupled with 3’ end processing?
Termination of transcription
How does alternate splicing permit multiple proteins to be produced by splicing defects?
- Exon removed – “skipping”
- Mutually exclusive exons – either or – never both
- Exon trunicaion/ extension at 5’ ends
- Exon trunication/ extension at 3’ ends
- Intron retained or removed
A mutation in what transcription factor causes Xeroderma pigmentosum (XP), and what are the symptoms?
TFIIH Photosensitivity Abnormal pigmentation Skin cancer susceptibility Neurological abnormalities Ability to perform DNA repaired (measured by UDS function) is 5-50% of normal
A mutation in what transcription factor causes Cockayne’s Syndrome (CS), and what are the symptoms?
TFIIH
Photosensitivity
Developmental defects
Neurological abnormalities
