Exon selection factor
U1 snRNP
Exon 1 AGGU
Factors such as U1 and
U2 snRNP identify splice sites
Intron 1
U2 snRNP
A AGG
Exon selection factor
Exon 2
Exons are identified by RNA sequences within the exons that are recognized by exon selection factors.

EXON1 INTRON1 PHENOTYPE
AG GT AGT CONSENSUS
G
GCCAG GTTGGTAT NORMAL
GCCAG A TTGGTAT

0 (no beta chains)
GCCAG T TTGGTAT

0 (no beta chains)
GCCAG GTTG
GCCAG GTTG
T
C
TAT
TAT

+ (some beta chains)

+ (some beta chains)
GCCAG GTTGG C AT

+ (some beta chains)

NORMAL: INTRON 1 EXON 2
TATTGGTCTATTTTCCCACCCTTAG GCTG 100%
MUTATION:
Normal site used 10% of the time: normal protein from these RNAs
TATT A GTCTATTTTCCCACCCTTAGGCTG
19 nucleotides
TATT A G TCTATTTTCCCACCCTTAGGCTG
New site used 90% of the time: no protein from these RNAs (note the shift in reading frame).
10%
90%
1
0
%
Net result: this allele shows a 90% reduction in β-globin production

A.
Normal stop codon is downstream or <50 bases upstream from splice junction
Translation
B.
Premature stop codon >50 bases upstream from splice junction
Last exon
Exon/Exon junction
>50 bases mRNA Decay
Last exon

Origin of premature stop codons
- Improper splicing
- intron retained
- frameshift
- Mutation
Possible consequences of premature stop codons:
- non functional protein
- formation of amyloid
- loss of a regulatory region from a protein that regulates growth
 cancer

Nucleus
Normal Stop Codon
Spliceosomes
Cytoplasm
Protein complexes
(Exon-junction-complexes; EJC)
Exon/Exon junctions
1st round of Translation
Complexes removed by ribosome transit
More translation

Spliceosomes
Nucleus
Premature stop codon
Normal stop codon
Cytoplasm
Protein complexes
(Exon-junction-complexes; EJC)
Exon/Exon junctions
1st round of Translation mRNA Decay
Complex not removed
Stopped ribosome

• Some drugs that affect the accuracy of codon recognition by ribosomes (such as gentamicin) decrease Nonsense mediated decay.
• Treatment with these drugs allows a low level of expression from genes with premature stop codons.
• Possible treatment for several disorders including some alleles of cystic fibrosis.
Reference: Holbrook et al Nature Genetics 36:801-808 (2004)

-30 start of transcription +30
TATA box
For most (but not all) promoters, a complex of proteins is assembled around the TATA box, located about 25-30 b.p. upstream from the start site. The consensus sequence of the
TATA box is TATAAA

Protein
DNA

-30 start of transcription +30
TATA binding protein
The TATA binding protein binds to the TATA box

-30 start of transcription +30
TFII-D
The TATA binding protein is one subunit of a large complex: TFII-D.

TFII-A
TFII-F
-30 start of transcription +30
TFII-B TFII-D
Several other complexes bind to TFII-D.

TFII-A
TFII-F
-30 start of transcription +30
TFII-B TFII-D
RNA pol II
RNA polymerase is recruited to the promoter.

TFII-A
TFII-F
-30 start of transcription +30
TFII-B
TFII-D
TFII-H
RNA pol II
The factor TFII-H plays a key role in initiating transcription by phosphorylating the C-terminal domain of the large subunit of RNA pol II.

CTD: The COOH Terminal Domain of the RNA pol II large subunit
…...(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)
52
COOH

CTD: The COOH Terminal Domain of the RNA pol II large subunit
…...(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)
52
COOH
ATP
TFIIH
ADP
…...(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)
52
COOH
TFIIH controls the start of transcription
Other kinases
PO
3 phosphorylated Ser 5 of the repeats
ATP
ADP
More phosphorylation of the CTD

CTD: The COOH Terminal Domain of the RNA pol II large subunit
Unphosphorylated CTD:
Involved in initiation:
Binding of initiation factors
Phosphorylated CTD:
Involved in elongation & RNA processing
Binds components involved in RNA capping
Binds components involved in RNA splicing
Binds components involved in 3’ end formation

The CTD ties elongation to capping, splicing and 3’-end formation
From Orphanadies & Reinberg (2002) Cell 108:439-51

NR NR
HRE
Fos Jun
TGACTCA
HAT
CREB CREB
GACGTC
Histone Acetyl Transferase
HRE (Hormone
Response Element)
Regulation by hormones such as estrogen which enter the cell
AP-1 (Fos-Jun binding site)
Regulation by growth factors, stress, and various transmembrane signals
CRE (Cyclic AMP
Response Element)
Regulation by cAMP, and by Ca + . Interacts with core promoter (through
CREB Binding Protein) and modifies chromatin structure (through HAT).
Positions of these elements are relatively unimportant
Core Promoter
Binds general transcriptional machinery
SP-1
GGGCGG
TATAAA
SP-1 Site
Provides basal unregulated transcriptional activity. Many genes have multiple
SP-1 sites

Phosphorylation of CREB and the CREB binding protein (CBP)
HAT
Serine 133

Signaling mediated by cAMP and protein kinase A
Hormone
Hormone receptor
Plasma membrane
G
Adenylate cyclase
ATP
G-protein
Inctive
Protein Kinase A cAMP Active
Protein Kinase A
Nuclear membranes
PO
4
PO
4
Active pKA enters the nucleus and phosphorylates
CREB on Serine 133
Core promoter
Phosphorylation of CREB:
- stimulates interactions with several core promoter proteins
- induces binding of HAT and acetylation of histones