Elongation phase: Transcription bubble

50 nt/sec

• unwound region 17 bp•RNA-DNA hybrid duplex 8 bp

•Mistakes 1 per 104- 1 per 104 nucleotides

Separation of RNA-DNA duplex

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

Two mechanisms possible

1) Rho () protein independent termination

stable hairpin formation

2) Rho () protein-mediated termination

G

GC

U

U

5'-U•C•C•C•A•C A•U•U•U-3'

G•C A•U C•G C•G G•C C•G C•G G•C

-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. 792-798Or 4th edition p.

Eukaryotic vs prokaryotic transcription

Prokaryotes:

• no membrane-bound nucleus

• transcription and translation are coupled

Eukaryotes:

• DNA is located in membrane-bound nucleus

• Transcription and translation are separated in space and time

Amanita phalloides (the death cap)

-amanitin

RNA Pol II Inhibitor

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 duplexand 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:

Examples:

Promoter sequenceStart site

Coding sequence

CAAT Box

(TATAAAA)

5'

-90 -25 +1

3'GC Box

-70

(GGGGCG)(GGNCAATCT)TATA box

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

NH

N

N O

NH2

N NN

H2N

O

HN

N

O

O

NN

N

N NH2

G•C A•T

NO

H

OH

N H

O

NN

O

Major groove

Minor groove

To deo

xyrib

ose

To deoxyribose

Formation of RNA Polymerase II pre-initiation complex

IIA stabilizes IID binding to promoter

IID contains TBP that binds TATA box

IIB binds initiation sequence

IIE stimulates transcription

IIH has kinase and helicase activity

Pol II binds IIB

Transcriptional control in eukaryotes

Coding region

Basal promoter

Upstream elements(CAAT, GC)

Regulated expression

Proximal elements(TATA box)

Enhancers

Enhancer sequences examples in eukaryotes

Post-transcriptional modifications of mRNA in eukaryotes

1. 5’ end CAP2. polyA tail3. 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