Transcript Nucleoside Phosphoramidate Monoesters: Potential

Elongation phase: Transcription bubble
Separation of
RNA-DNA duplex
50 nt/sec
• unwound region 17 bp
•RNA-DNA hybrid duplex  8 bp
•Mistakes 1 per 104- 1 per 104 nucleotides
RNA Synthesis in Bacteria
Start of Gene
Several RNA Pol transcribing the
same gene
In bacteria, the new RNA can be immediately
translated
Termination of RNA Polymerization
U
Two mechanisms possible
1) Rho () protein independent termination
C
U
G
G
G•C
A•U
C•G
C•G
G•C
C•G
C•G
G•C
stable hairpin formation
5'-U•C•C•C•A•C
2) Rho () protein-mediated termination
A•U•U•U-3'
-independent termination
-dependent termination
Antibiotic inhibitors of RNA
polymerization
Rifampicin inhibits initiation by blocking a channel within RNA polymerase:
Actinomycin intercalates in ds DNA, inhibiting strand separation:
phenoxazone
RNA synthesis in Eukaryotes
See Stryer 5th edition p. 792-798
Or 4th edition p.
Eukaryotic vs prokaryotic transcription
Prokaryotes:
Eukaryotes:
• no membrane-bound nucleus
• DNA is located in membranebound nucleus
• transcription and translation
are coupled
• Transcription and translation are
separated in space and time
a-amanitin
RNA Pol II Inhibitor
Amanita phalloides (the death cap)
Actinomycin D
•Antitumor antibiotic from Streptomyces genus
• Aromatic ring intercalates between GC base pairs, while the peptides
bind to the minor groove
• Binds to GpC sequences in double-stranded DNA, stabilizing the duplex
and inhibiting transcription
• Inhibitor of eukaryotic RNA Pol I
Eukaryotic RNA Polymerase II
RNA Pol II is responsible for transcription to pre-mRNA
-
8-12 subunits
-
Two large subunits are responsible for RNA synthesis
-
Regulated by phosphorylation of carboxyl-terminal domain
(CTD) of the largest subunit
Unphosphorylated form is involved in initiation and
phosphorylated form in elengation
-
some subunits are shared for RNA Pol I-III
Type II Eukaryotic Promoters
Consensus sequence:
-90
5'
CAAT Box
(GGNCAATCT)
-70
GC Box
(GGGGCG)
Promoter sequence
Examples:
-25
+1
3'
TATA box
(TATAAAA)
Coding sequence
Start site
Initiation of transcription in eukaryotes
• RNA Pol II can not initiate transcription by
itself
• Transcription factors (TFII) are required
• The key initiation step is the recognition of
TATA box by TBP
TATA box-binding protein (TBP)
Hydrogen bond donors and acceptors on each edge of a base pair
M ajo r g ro o v e
T
o
d
x
eo
i
yr
s
bo
e
To
M inor groove
de
ox
yr
ib
os
e
Formation of RNA Polymerase II pre-initiation complex
IID contains TBP that binds TATA box
IIA stabilizes IID binding to promoter
IIB binds initiation sequence
Pol II binds IIB
IIE stimulates transcription
IIH has kinase and helicase activity
Transcriptional control in eukaryotes
Enhancers
Upstream elements
(CAAT, GC)
Regulated expression
Proximal elements
(TATA box)
Basal promoter
Coding region
Enhancer sequences examples in eukaryotes
Post-transcriptional modifications of
mRNA in eukaryotes
1. 5’ end CAP
2. polyA tail
3. splicing
Eukaryotic mRNA is 5’-Capped
PolyA Synthesis and termination of
transcription
RNA splicing in eukaryotes
Primary transcript,
hnRNA
RNA Synthesis: Take Home Message
1) DNA sequences are translated into RNA
messages by RNA polymerases.
2) The initiation of RNA synthesis is controlled
by specific DNA promoter sequences.
3) The synthesis of RNA is governed by
initiation, elongation, and termination steps.
4) Eukaryotic mRNA is extensively processed