Transcription Flashcards
Transcription is ___-directional and synthesizes nucleotides in a __—->__ fashion.
uni-directional; 5’ –> 3’
What are the five basic sterps of transcription?
- RNA Pol binds the promoter on closed DNA
- RNA Pol melts DNA forming transcription bubble, aka open complex
- Pol catalyzes linkage of first two rNTPs
- Pol advances down DNA 3’->5’ linking and melting
- Pol releases RNA and falls off DNA at transcription stop site
Describe the 4 cellular RNA polymerases
Bacterial RNA Pol: does everything
Human Pol I: rRNA
Pol II: mRNA, snRNA, miRNA. It has CTD!!!
Pol III: tRNA
Define a promoter and give an example
Sequence upstream of a gene that positively impacts transcription. TATA box 30bp upstream
How does alpha-amanitin work?
It inhibits the movement of RNA Pol II, binding its bridge substructure so that translocation of the polymerase down the DNA chain can’t happen.
How does rifampicin work?
Binds the beta subunit of bacterial RNA Pol; plugs up the exit chamber where assembled RNA exits the transcriptional complex.
Describe structures of the Pre-Initiation Complex.
TFII-A,B,D,E,F,H, Pol II, and Med
TFIID = TBP (TATA Binding Protein)
TFIIH = Helicase and phosphorylates CTD
Describe the clinical syndromes caused by mutations in TFIIH subunits.
Problems with NER: Cockayne’s syndrome, Trichothiodystrophy, Xeroderma pigmentosum - light sensitivity, abnormal pigmentation, cancer susceptibility, neuorological abnormalities, unscheduled DNA synthesis
Describe the three major ways in which most pre-mRNA’s are processed.
- The first 5’ triphosphate is replaced with a backwards, 7-methylguanosine
- Splicing: excision of introns and desegmentation of exons
- Cleavage/Polyadenylation: cleavage of RNA at 3’ end past the consensus sequence and polyadenylation (> 200 A’s) of cleaved site.
List the 3 functions of the 5’ cap of the mRNA.
- Prevents degradation by exonucleases (removal signals degradation)
- Facilitates splicing & processing via CBC
- Recognized by eIF4E for transport to ribosomes
List the three reactions required to add a 5’ cap to pre-mRNA
- Cut off PO4 at 5’ end
- Add GTP backwards via Guanylyl Transferase (It becomes GMP). Keep 2 PO4 from mRNA
- Methylate the 7-position of guanosine cap via SAM. Thus, 7-methyl-guanosine cap
What conserved sequences are at the 5’ and 3’ ends of most introns?
Splice site at 5’ end of intron: GU (beginning)
Splice site at 3’ end of intron: AG. (last 2 bases)
What is the consensus sequence at poly-A site?
AAUAAA
Describe how alternative splicing permits multiple proteins to be produced by splicing defects.
Remove/retain certain exons mutually exclusive exons exon truncation or extension at 5' end exon truncation or extension at 3' end intron retained
How can alternative splicing go awry?
The 5’ splice site at any intron is still part of the DNA, and is therefore vulnerable to mutation
Provide two examples of genetic disorders caused by splicing defects.
Marfan’s syndrome: disruption of splicing the fibrilin gene transcript (fibriling = conn. tissue in wall of heart and blood vessels). Tall people prone to aneurysms.
CD44: abnormal splicing is a predictor of tumor metastasis (diag. & prog. marker)
Describe the function of U1 and U2 snRNA’s in splicing
U1: binds GU
U2: binds branch point (A)
U2AF: binds AG
Lariat mechanism where U1 brings 5’ into proximity with branch point. U2 activates the 2’ OH at branch point, which attacks the phosphodiester bond just past GU. Now, the free 3’ OH on the end of the 5’ exon attacks the phosphodiester bond at the AG 3’ splice site, linking the two exons and excising the snRNP/intron complex to be degraded
Where are the 5’ and 3’ UTR sequences
5’: between +1 and first Start Codon (XUG)
3’: after stop codon (UAA, UAG, UGA) through poly-A tail
Describe the two reactions that make the mature 3’ end of mRNA’s.
- Recognition and cleavage of pre-mRNA after AAUAAA
2. Polyadenylation of 3’ end
The poly(A) tail is important for what 3 things?
nuclear export, translation, and stability of mRNA.
What event terminates transcript?
3’ end processing (cleavage/polyadenylation)
Provide an example of how alternative poly A sites can be used to make more than one protein from a single gene.
Just like alt. splicing, it depends on where the poly A is added. Example: two forms of IgM (memb-bound and secreted) are formed by alt poly-A sites in their common gene.
