Transcription Flashcards
How are phosphodiester bonds formed by RNA polymerase?
3’OH attacks alpha phosphate attached to 5’ carbon (releases 2 P groups)
5 steps in transcription cycle
Initiation, Elongation, Termination
1) RNA pol binds promoter sequence on helical DNA in a closed complex
2) Pol melts DNA strands apart near transcription start site forming an open complex
3) Polymerase catalyzes phosphodiester linkage of two initial rNTPs
4) Polymerase advances 3’–> 5’ down template strand, linking rNTPs 5’–>3’
5) At transcription STOP site, polymerase releases completed RNA and dissociates from DNA
RNA pol I
makes rRNA
RNA pol II
- makes mRNA, snRNA, microRNA
- CTD
- Needs general transcription factors (TFIIH, TFIID)
- Uses sliding clamp
C-terminal Domain (CTD)
- on RNA pol II
- acts as a landing pad for proteins that effect elongation/processing
RNA pol III
makes tRNA
Promoter
- sequence of DNA upstream of the transcription start site that direct RNA polymerase to the start of genes
- controls FREQUENCY of transcription
- site of pre-initiation complex assembly
TATA Box
- promoter sequence (TATAAA)
- 30 bases upstream from start site
- conserved
- site where GTFs bind
TATA box binding protein
- binds TATA box
- directs assembly of pre-initiation complex of general transcription factors at the promoter
+1 base
-) and (+
-first base pair where transcription initiates
(-) = upstream (+) = downstream
Proximal Promoter Elements
- gene specific sequence that specific TFs bind
- Influence activity of the promoter
Enhancer
- either up or down stream
- can be very far away
- bind transcriptional activator proteins and effect transcription
- cell type specific
a-amanitin (and transcription)
- non-competitive inhibitor of RNA polymerase
- Binds bridge helix of RNA pol II blocking translocation
Rifampicin (and transcription)
- Inserts itself into RNA exit channel of bacterial RNA polymerase
- Block growth of the RNA chain
TFIID
- part of RNA polymerase II pre-initiation complex
- aka TATA binding protein
- promoter recognition GTF
- binds TATA box on DNA sequence
TFIIH
- part of RNA polymerase II pre-initiation complex
- promoter recognition GTF
- Functions in transcription and DNA repair (nucleotide excision)
- has multiple subunits (CDK7 protein kinase and XPB helicase)
CDK7 Protein Kinase
- part of TFIIH
- phosphorylates RNA pol II CTD during promoter clearance
- Allows pol II to elongate
XPB helicase
- part of TFIIH
- acts as helicase to open up DNA strands
Syndromes associated with mutations in TFIIH (3)
- Xeroderma Pigmentosum (XP)
- Cockayne’s Syndrome (CS)
- Trichothiodystrophy (TTD)
Xeroderma Pigmentosum (XP)
mutation in TFIIH so that is is unable to carry out nucleotide excision repair for damage caused by UV light
3 ways pre-mRNAs are processed
- Capping
- Splicing
- Cleavage/Polyadenylation
Capping
replacement of 5’triphosphate from the first rNTP with a backwards 7-methylguanosine (no phosphate group)
Splicing
excision of introns, desegmentation of exons
Cleavage/Polyadenylation
cleavage of RNA at 3’end past consensus sequence, and polyadenylation (addition of > 200A’s) of cleaved site
Pre-mRNA
- initial product of transcription that must be extensively processed into a mature form (mRNA)
- has introns AND exons
- no cap, no tail
Processing into mRNA enhances _________, _________, and ___________
nuclear export, translation, preventing degredation
Function of 5’cap of mRNA (4)
- Protects the 5’end from exonucleases
- Recognized by cap-binding proteins - crucial for splicing/processing
- Recognized by TF eIF4E for nuclear export into cytoplasm
- Removal of cap signals mRNA degredation
3 reactions required to add a 5’ cap to pre-mRNA
1) Triphosphatase - removes gamma phosphate at end of primary transcript
2) Guanylytransferase - synthesizes 5’–>5’ phosphodiester linkage
3) Guanine 7-methyltransferase - addition of methyl group at the 7 position
Splice site at 5’ end of intron
GU
Splice site at 3’ end of intron
AG
Consensus sequence at poly-A site
AAUAAA
Alternative splicing
allows many different proteins to be encoded by a single gene
Genetic disorders caused by splicing defects (2)
1) CD44 - tumor metastasis
2) Spinal muscular atrophy
CD44 and tumor metastasis
abnormal splicing of CD44 contributes to tumor metastasis
Spinal muscular atrophy treatment
can activate alternate SMN2 gene to take over for defective SMN1 gene by using alternative splicing
HIV and alternative splicing
HIV uses alternative splicing to generate many more mRNAs from a single transcript
Steps of splicing (4) (trans-esterification reactions)
1) U2snRNA base pairs with branch point A, and activates it
2) U1 snRNA basepairs with 5’ splice site
3) 2’OH of branch point A then attacks phosphodiester bond IN FRONT of GU marker ath beginning of intron
4) 3’OH of exon 1 attacks phosphodiester bond at AG splice site, linking exon 1 and exon 2
U2 snRNA
- key role in splicing reactions
- base pairs with branch point A and activates it
U1 snRNA
- key role in splicing reactions
- base pairs with 5’ splice site
Transcription start site
+1 position
Introns
non-coding sequences between GU (5’) and AG (3’)
branch point
- single A residue
- usually in intron between GU (5’) and AG (3’)
- base pairs with U2 snRNA (VITAL part in first step of splicing)
Exons
coding sequences
5’UTR
- untranslated region
- between +1 and start codon on the processed mRNA strand
- located at beginning of first exon
3’UTR
- untranslated region
- located after stop codon, at the end of the last exon
- contains highly conserved polyA site
initiation/start codon
- 5’AUG
- encodes methionine
termination/stop codon
3’ UAG, UAA, UGA
polyA site
consensus sequence AAUAAA
Two reactions that make mature 3’ end of mRNAs
1) Cleavage: recognize consensus sequence (AAUAAA) at 3’ end of pre-mRNA and cleave
2) Polyadenylation: polyadenylation of free 3’ hydroxyl group
3’ end processing of pre-mRNA is coupled with ________________ because ____________
- termination of transcription by RNA pol II
- because 3’ polyA tail necessary for RNA polymerase complex to detach from the DNA
Function of mRNA polyA tail (2)
1) protection from degradation
2) Export of mRNA from the nucleus
Mutations in polyA site can result in _________
Thallasemia
Thallasemia
results from mutations of the AAUAAA poly A site consensus sequence in globin genes
IgM (immunoglobulin M) and alternative poly A sites
Membrane bound and secreted forms are formed based on alternative polyA sites in their common genome
Cancer cells and alternative poly A sites
use altered polyA site choice to shorten the 3’UTR region, allowing them to escape regulation by microRNAs
