Transcription, RNA Processing (Hampsey) Flashcards
Rifampin in binds to
Bacterial RNAP
Prokaryotic RNAP consists of
Sigma subunit, associates with core enzyme (alpha, alpha, beta, beta, omega) to generate hold enzyme
What happens during prokaryotic transcription
1. Initiation
Holo-RNAP (sigma) binds promoter, principal site for regulation of transcription
What happens during prokaryotic transcription: Elongation
RNAP “leaves the station”; sigma dissociates from RNAP & promoter
What happens during prokaryotic transcription: Termination
DNA-RNA-RNAP complex disassembles..dependent on specific signals (Rho dependent or Rho independent)
Structure of a bacterial promoter, how many sigma binding sites?
2
-35 sequence (TTGACA)
Pribnow box (TATAAT)
**these consensus sequences are not exact, don’t want overly tight binding
Rho-dependent transcription termination
Travels along nascent mRNA, comes in contact with RNAP and dissociates complex to stop termination
*torpedo, ATP dependent
Rho-independent transcription termination
- Stem and loop
- mRNA folds up and makes this stem
- still within RNAP and complex disassociates
- 3’ end: UUUU-OH
True or false: there are DNA sequences that tell transcription to stop
TRUE…..Rho-dependent/independent transcription signals are encoded for by DNA
2 notable features of eukaryotic transcription
1) 5’-CAP
2) 3’ polyA tail
Alpha-amantin
- eukaryotic transcription inhibitor
- binds RNAP III and inhibits elongation (just like rifampicin does this in bacteria)
DNA –> rRNA via
RNAP I
DNA–> mRNA via
RNAP II
DNA–>tRNA/ssRNA via
RNAP III
MRNA—>Protein via
Ribosome
These bind to regulatory proteins and activate stuff…
>250bp
-dsDNA, function in any direction, and are binding sites for transcription activators
Enhancers
CpG islands make up
Proximal promoter
-bind regulatory proteins for housekeeping genes & certain activator proteins
Ex: Sp1: most well characterized activator for proximal promoter
Core promoter is the
TATA box
-40 to +40
Transcription start site +1 (INR, DPE is +20)
Binding site for RNAP II and GTFs
What makes up core promoter
BRE (TFIIB recognition element) @ -35
TATA @ -25INR +1
DPE +30
Proximal promoter and Core promoter….
Span ~80 bp & flank start of transcription
What binds to dsDNA Pre initiation complex
GTFs
—>leads assembly of RNAP II
Recognize specific stuff in promoter, similar to sigma in bacteria
What positions RNAP II at the promoter
GTFs (they recognize promoter elements)
GTFs specific to RNAPII
TFIIA, TFIIB, TFIID
TFII B binds to
BRE of core promoter
TFII D binds to
Promoter
- includes TBP (TATA binding protein) + 14 TBP associated factors (TAFs)
- nucleases assembly of transcription Pre initiation complex
> > > > TBP (part of TFDII) binds TATA > bend in promoter DNA> allows TFBII and other to bind»»TFIIE,TFIIH bind subsequently, involved in promoter melting so RNAP II transcription can begin
Which GTF is the last to bind?
TFII H
-has helicase activity and can unwind DNA, then DNA can descend into active site of RNAPII
TFIIH implicated in
XP (xeroderma pigmentosum)
TTD (trichotriodystrophy)
Cockade syndrome (CS)
Many activators can stimulate transcription by binding GTFs in order to
Recruit or stabilize their binding to core promoter
Where is the only place where 5’ to 5’ linkage occurs
7-methylguanosine cap at 5’ ends of mRNAs
- via triphosphate linkage
- as soon as mRNA emerges from RNAP it’s capped 4 protection
What occurs during polyadenylation of a primary transcript
There is a cleavage signal: AAUAA, and 2 enzymatic reactions
1) cleavage by specific endnonuclease
2) addition of tail by polyApolymerase (not encoded in DNA, uses ATP)
Why is polyadenylation of a primary transcript needed?
For mRNA stabilization, transport of mRNA from nucleus to cytoplasm
-uses ATP
Direction of flow of genetic information
5’ to 3’
What’s the organization of a human B-glob in gene?
3 exonuclease, 2 introns
-before RNA translated, splicing occurs
How is b-glob in gene processed?
Bglobin gene-[transcription, 5’ cap, poly a tail]->primary transcript (with introns)-[splicing]->B globin mRNA
5’ splice site of upstream exon
AG|GUAAGU
First exon always begins with a GU
3’ splice site of downstream exon
CAG|G
***exon always ends with AG
Exon begins/ends with
GU…….AG
Where is there the only 2’ to 5’ PD bond?
Lariat intermediate (which eventually becomes intron)
Transesterification reaction
1) cleave PH bond btwn exon 1, intron 1 (2’OH branch site of intron) —->form the 2’-5’ PD bond
2) 3’-OH of exon 1 cleaves PD bond btwn intron, exon 2
- –>exon 1 joined to exon 2, intron released as lariat
Spliceosome assembly is de novo from _____
SNURPs
SNURPs recognize
5’ splice site (AG|GU) U1
Branch sit
3’ splice site(AG|G)
Branch site U2
+U4U5U6 complex –>basically scrunched it up
Spliceosome assembly requires
ATP
B-thalassemia is an intron mutation..what kind?
Point mutation (substitution) -now machinery thinks this is splice site and have 6 extra amino acids as a consequence
Tamoxifen
- antagonist
- binds to estrogen receptor as a competitive inhibitor
- fails to induce conformational change required for transcription activation
How do steroid hormones work?
Directly stimulate transcription as hormone-receptor complexes…receptors are transcription factors; bind to dsDNA (promoter) and can cause conformational changes
DNA-Hormone-Receptor Comoplex interacts with coactivator complex –> RNAP II transcriptional machinery stimulated
Steroid hormones bind ____ and recruit chromatin remodeling proteins
Enhancer sequences
How do coactivators work?
Recruit/stabilize RNAP II/GTF to core promoter
-clear a path for RNAP II by altering chromatin DNA template
By remodeling chromatin, can now recruit coactivator complex (how estrogen works)
What do histone acetyltransferases (HATs) do?
Acetylation of histone lysine residue in N-terminal tails of H3, H4 –>chromatin modification
*reversible by histone deactylases (HDACs)
ATP-dependent remodeling complexes (aka remodeling engine) does what?
Remodel nucleosomes via ATP hydrolysis
–> TFs bind to DNA and promote gene transcription
Mechanism of glucocorticoid receptor
Cortisol binds -> conformational Chang win receptor -> zinc finger binding domain uncovered -> steroid receptor complex interacts with specific regulatory DNA sequences (GRE)
-hormone receptor complex (w/ coactivator proteins) controls transcription of targeted genes
MMTV (mouse mammaery tumors virus)
-DNA virus
- breach cell carcinoma
- MMTV has DNA sequence identical to estrogen enhancer, integrates next to Wnt-1-protooncogene so WNt1 now regulator of cell proliferation under control of estrogen
TFIIH
Couples transcription with DNA damage repair
Issue with TFII H associated with some is eases (XP,TTD,CS) since DNA rapier altered
Alpha amantin
Binds to RNAP II , inhibits elongation of transcription
Rifampicin
Antibiotic,binds to RNAP of bacteria
