TRANSCRIPTION

LECTURES, FALL 2010

NABIL BASHIR
RNA Structure and Transcription- Prokaryotes
a). Chemistry of RNA
i). Bases found in RNA
ii). Ribose sugar
iii). RNA polynucleotide chain
iv). Secondary and tertiary structure
b). Characteristics of prokaryotic RNA
i). Classes of prokaryotic RNA
ii). Structure of prokaryotic messenger RNA
c). Transcription initiation in prokaryotes
i). Transcription
ii). Promoter structure
iii). Prokaryotic RNA polymerase structure
iv). Initiation of transcription and the sigma cycle
 Learning Objectives
• Compare and contrast the chemistry of DNA and
RNA
• Know the major classes of RNA in prokaryotes
• Understand the structure of prokaryotic mRNA
• Understand the structure of the prokaryotic
promoter
• Understand the structure of bacterial RNA
polymerase and know the class of antibiotics
that inhibits this enzyme
• Understand the function of the sigma factor in
the initiation of transcription in E. coli
 The major bases found in DNA and RNA

DNA RNA

Adenine Adenine
Cytosine Cytosine
Guanine Guanine
Thymine Uracil (U)

thymine-adenine base pair uracil-adenine base pair

 Examples of modified bases found in RNA

Dihydrouridine Pseudouridine 1-methylguanosine 7-methylguanosine

1-methyladenosine 2-thiocytidine 5-methylcytidine Ribothymine

 RNA polynucleotide chain
• 2’ -OH makes
3’, 5’ phosphodiester
bond unstable

DNA polynucleotide chain

Secondary structure

Tertiary structure
Classes of prokaryotic RNA
• ribosomal RNA (rRNA)
16S (small ribosomal subunit)
23S (large ribosomal subunit)
5S (large ribosomal subunit)
• transfer RNA (tRNA)
• messenger RNA (mRNA)

Structure of prokaryotic messenger RNA

Shine-Dalgarno sequence initiation
5’ PuPuPuPuPuPuPuPu AUG
translated region
3’ AAU
termination

The Shine-Dalgarno (SD) sequence base-pairs with a pyrimidine-rich

sequence in 16S rRNA to facilitate the initiation of protein synthesis
 Transcription
closed promoter complex

RNA polymerase
open promoter complex

initiation

elongation

termination

RNA product
Promoter structure in prokaryotes

mRNA
5’ PuPuPuPuPuPuPuPu AUG
-30 -10 +1
[ ]
Promoter
transcription start site
mRNA
5’
-30 region -10 region
TTGACA TATAAT
AACTGT ATATTA
-36 -31 -12 -7 +1 +20
Pribnow box
T T G AC A TATA AT
82 84 79 64 53 45% 79 95 44 59 51 96%

consensus sequences
Prokaryotic RNA polymerase structure

RNA polymerase of bacteria is a multisubunit protein

Subunit Number Role

a 2 uncertain
b (Rifampicin target) 1 forms phosphodiester bonds
b’ 1 binds DNA template
s 1 recognizes promoter and
facilitates initiation

a2bb’s a2bb’ + s
holoenzyme core polymerase sigma factor
 The function of sigma factor
• the sigma subunit of RNA polymerase is an “initiation factor”
• there are several different sigma factors in E. coli that are
specific for different sets of genes
• sigma factor functions to ensure that RNA polymerase binds
stably to DNA only at promoters
• sigma destablizes nonspecific binding to non-promoter DNA
• sigma stabilizes specific binding to promoter DNA
• this accelerates the search for promoter DNA
Ka (M-1)
Any DNA Promoter DNA
(nonspecific) (specific)

Core 2 X 1011

Holo 1 X 107 1013 to 1015

• promoters vary in “strength” by ~two orders of magnitude

 • closed promoter complex (moderately stable)

s
• the sigma subunit binds to the -10 region

RNA polymerase holoenzyme (+ s factor)

• open promoter complex (highly stable)

• the holoenzyme has very high affinity for

s
promoter regions because of sigma factor

• once initiation takes place, RNA polymerase does

not need very high affinity for the promoter
• sigma factor dissociates from the core polymerase
after a few elongation reactions

s
• elongation takes place with
the core RNA polymerase

• sigma can re-bind

other core enzymes The sigma cycle
 Mechanism of RNA synthesis
RNA RNA

A=T A = T

U=A U=A

• RNA synthesis usually initiated with ATP or GTP (the first nucleotide)
• RNA chains are synthesized in a 5’ to 3’ direction
 Eukaryotic Transcriptional Regulation

a). Characteristics of eukaryotic RNA and their polymerases

i). Classes of cellular RNA
ii). RNA polymerases I, II, and III
b). Transcription of messenger RNA in eukaryotes
i). Structure of eukaryotic messenger RNA
ii). Complexity of mRNA populations in the cell
iii). Promoters and transcription elements
iv). Transcription factors
General transcription factors
Basic region-leucine zipper proteins
Zinc finger transcription factors
v). Mutations affecting promoters
 Learning Objectives
• Know the major classes of RNA in eukaryotes, their RNA polymerases, and
what inhibits RNA polymerase II
• Understand the structure of eukaryotic mRNA
• Understand the structure of the eukaryotic promoter
• Understand the fact that mRNAs exist in different abundance classes and
that these differences are due largely to transcriptional regulation
• Understand how the preinitiation complex forms
• Understand the role of transcription factors and how they bind transcription
response elements in DNA
• Understand the structure and function of the bZIP transcription factors
• Understand the structure and function of the zinc finger transcription factors
belonging to the nuclear receptor superfamily of transcription factors
• Understand how mutations can affect the function of the factor IX promoter
 Classes of eukaryotic cellular RNAs

• ribosomal RNA (rRNA)

18S (small subunit)
28S (large subunit)
5.8S (large subunit)
5S (large subunit)
• transfer RNA (tRNA)
• messenger RNA (mRNA)
• heterogeneous nuclear RNA (hnRNA) (precursors of mRNA)
• small nuclear RNA (snRNA)
U1, U2, U3, U4, U5, U6, U7, U8, U9, U10...
• small cytoplasmic RNA (scRNA)
7SL RNA

What are the enzymes responsible for the synthesis of these RNAs?
 The human RNA polymerases
Polymerase Location Product

RNA polymerase I nucleolus 18S, 28S, 5.8S rRNA

RNA polymerase II nucleoplasm hnRNA/mRNA,
U1, U2, U4, U5 snRNA
RNA polymerase III nucleoplasm tRNA, 5S RNA,
U6 snRNA, 7SL RNA
mitochondrial
RNA polymerase mitochondrion all mitochondrial RN
_____________________________________________________________________________________________

Sensitivity of the nuclear RNA polymerases to a-amanitin1

RNA pol I resistant
RNA pol II high sensitivity (binds with K = 10-8 M)
RNA pol III low sensitivity (binds with K = 10-6 M)
1
cyclic octapeptide from the poisonous mushroom Amanita phalloides
 Structure of eukaryotic mRNA

Cap 5’ untranslated region initiation

5’ 7mGpppN AUG

translated region
UGA
termination
3’ untranslated region

polyadenylation signal
AAUAAA (A)~200 3’
poly(A) tail
• all mRNAs have a 5’ cap and all mRNAs (with the exception
of the histone mRNAs) contain a poly(A) tail
• the 5’ cap and 3’ poly(A) tail prevent mRNA degradation
• loss of the cap and poly(A) tail results in mRNA degradation
Complexity1 of mRNA classes in the mammalian cell2
Number of
different
Abundance Abundance mRNA
class (copies/cell) species Total
high 12,000 9 108,000
intermediate 300 700 210,000
low (rare) 15 11,500 172,500
12,209 490,500
Based on these measurements, this cell type contains
• three abundance classes of mRNA
• ~ 12,209 different mRNA species
• ~490,500 total mRNA molecules
1
determined in RNA-DNA hybridization experiments analogous to Cot curves
2
mouse liver cytoplasmic poly(A)+ RNA
• how are these mRNAs made and what determines their relative amounts?
• rate of synthesis vs. rate of turnover (degradation)
 Transcription and promoter elements for RNA polymerase II

transcription
+1 transcription unit
element
TE P exon exon
promoter
Promoter (DNA sequence upstream of a gene)
• determines start site (+1) for transcription initiation
• located immediately upstream of the start site
• allows basal (low level) transcription
Transcription element (DNA sequence that regulates the gene)
• determines frequency or efficiency of transcription
• located upstream, downstream, or within genes
• can be very close to or thousands of base pairs from a gene
• includes
enhancers (increase transcription rate)
silencers (decrease transcription rate)
response elements (target sequences for signaling molecules)
• genes can have numerous transcription elements
Transcription and promoter elements for RNA polymerase II

transcription
element transcription unit
TE P exon exon
promoter
transcription
element
TE P exon exon
promoter complex

TE P exon exon TE

P exon TE exon
The locus control region is a specialized transcription element

locus control region

LCR

TE
gene A
P

gene B
TE P

• a single locus control region (LCR) may control two or more

transcription units in a cell-specific fashion
 Sequence elements within a typical eukaryotic gene 1
1
based on the thymidine kinase gene
octamer
transcription
+1
promoter
element
ATTTGCAT GC CAAT GC TATA
-130 -95 -80 -50 -25

TATA box (TATAAAA)

• located approximately 25-30 bp upstream of the +1 start site
• determines the exact start site (not in all promoters)
• binds the TATA binding protein (TBP) which is a subunit of TFIID
GC box (CCGCCC)
• binds Sp1 (Specificity factor 1)
CAAT box (GGCCAATCT)
• binds CTF (CAAT box transcription factor)
Octamer (ATTTGCAT)
• binds OTF (Octamer transcription factor)
 Proteins regulating eukaryotic mRNA synthesis

General transcription factors

• TFIID (a multisubunit protein) binds to the TATA box
to begin the assembly of the transcription apparatus
• the TATA binding protein (TBP) directly binds the TATA box
• TBP associated factors (TAFs) bind to TBP
• TFIIA, TFIIB, TFIIE, TFIIF, TFIIH1, TFIIJ assemble with TFIID

RNA polymerase II binds the promoter region via the TFII’s

Transcription factors binding to other promoter elements and

transcription elements interact with proteins at the promoter
and further stabilize (or inhibit) formation of a functional
preinitiation complex
1
TFIIH is also involved in phosphorylation of RNA polymerase II, DNA repair
(Cockayne syndrome mutations), and cell cycle regulation
Binding of the general transcription factors

E F TAFs

B TFIID
H

TBP
A J
-25 +1
• TFIID (a multisubunit protein) binds to the TATA box
to begin the assembly of the transcription apparatus
• the TATA binding protein (TBP) directly binds the TATA box
• TBP associated factors (TAFs) bind to TBP
• TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, TFIIJ assemble with TFIID
Binding of RNA polymerase II

E F

B TFIID
H

TBP
A J
RNA pol II

• RNA polymerase II (a multisubunit protein) binds to

the promoter region by interacting with the TFII’s
• TFs recruit histone acetylase to the promoter
Binding of specialized TFs

E F

B TFIID
H

TBP
A J +1
RNA pol II
• transcription factors binding to
other promoter elements and transcription elements interact
with proteins at the promoter and further stabilize (or inhibit)
formation of a functional preinitiation complex
• this process is called “transactivation”
 Formation of a stable preinitiation complex

E F

B TFIID H

TBP
J +1
RNA pol II

• the stability and frequency with which complexes are formed

determines the rate of initation of transcription
• the rate of initiation of transcription is of major importance in
determining the abundance of an mRNA species
 Initiation of transcription and promoter clearance

E F

B TFIID H initiation
TBP
J +1 RNA pol II

P CTD

P
P
• RNA pol II is phosphorylated by TFIIH on the carboxy terminal
domain (CTD), releasing it from the preinitiation complex and
allowing it to initiate RNA synthesis and move down the gene
 Transcription factors (partial list)
Factor Full name or function
CREB Cyclic AMP response element binding protein
CTF CAAT box transcription factor (=NF1) (binds GGCCAATCT)
NF1 Nuclear factor-1 (=CTF)
AP1 Activator protein-1 (dimer of the Fos-Jun proteins)
Sp1 Specificity factor-1 (binds CCGCCC)
OTF Octamer transcription factor (binds ATTTGCAT)
NF-kB Nuclear factor kB
HSTF Heat shock transcription factor
MTF Metal transcription factor
USF Upstream factor
ATF Activating transcription factor
HNF4 Hepatocyte nuclear factor-4 (nuclear receptor superfamily)
GR Glucocorticoid receptor (nuclear receptor superfamily)
AR Androgen receptor (nuclear receptor superfamily)
ER Estrogen receptor (nuclear receptor superfamily)
TR Thyroid hormone receptor (nuclear receptor superfamily)
C/EBP CAAT/enhancer binding protein
E2F E2 factor (named for the adenovirus E2 gene)
p53 p53 (tumor suppressor protein)
Myc Product of the c-myc protooncogene (dimerizes with Max)
Basic region-leucine zipper (bZIP) transcription factors

• Leucine zipper functions in dimerization

• Basic region binds DNA within the major groove

• Example of a bZIP transcription factor:

• AP1 (Fos-Jun or Jun-Jun dimers)

• The Fos and Jun families each contain several different
proteins that can homo- or heterodimerize
• Fos and Jun are products of the fos and jun protooncogenes
• AP1 is involved in the regulation of gene expression as
controlled by various growth factors, hormones, tumor
promoters, neuronal stimulation, and cellular stress
The Fos and Jun proteins

Fos Jun
Basic regions
(DNA contact surfaces
that bind to the DNA)

• Four leucines ( ) are present Leucine zipper

at every seventh position in (dimerization
the amphipathic a-helix domain)
Helical wheel analysis of a leucine half-zipper
Leucine at every seventh position

Amphipathic alpha helix

Dimerization of the AP1 transcription factor N

N
DNA binding
• Dimerization via the leucine domains

zippers brings together the

DNA binding domains of the
two proteins, providing a
sufficient amount of binding
surface to form a stable
protein-DNA interaction
• The leucine zippers interact via their
dimerized
hydrophobic faces forming coiled coils leucine
that cause the two proteins to dimerize zippers

C C
Gcn4 (Basic Region, Leucine Zipper) Complex With Ap-1 DNA
Structures generated using RasWin Molecular Graphics
Windows Version 2.6 and PDB ID# 1YSA

DNA binding

Leucine zipper
Binding of AP1 to DNA transactivates transcription

E F

B TFIID
H
TGACTCA TBP
ACTGAGT A J +1
RNA pol II

• Binding of AP1 to its

DNA transcription element (TGACTCA) stimulates
RNA synthesis by interacting with the preinitiation complex
Binding of AP1 to DNA transactivates transcription

E F

P
Fos Jun B TFIID
H
TGACTCA TBP
ACTGAGT A J +1
RNA pol II

• Binding of AP1 to its DNA transcription element (TGACTCA)

• Activity of AP1 can be further regulated by phosphorylation
by Jun N-terminal kinase (JNK “junk” kinase)
 Transcription factors (partial list)
Factor Full name or function
CREB Cyclic AMP response element binding protein
CTF CAAT box transcription factor (=NF1) (binds GGCCAATCT)
NF1 Nuclear factor-1 (=CTF)
AP1 Activator protein-1 (dimer of the Fos-Jun proteins)
Sp1 Specificity factor-1 (binds CCGCCC)
OTF Octamer transcription factor (binds ATTTGCAT)
NF-kB Nuclear factor kB
HSTF Heat shock transcription factor
MTF Metal transcription factor
USF Upstream factor
ATF Activating transcription factor
HNF4 Hepatocyte nuclear factor-4 (nuclear receptor superfamily)
GR Glucocorticoid receptor (nuclear receptor superfamily)
AR Androgen receptor (nuclear receptor superfamily)
ER Estrogen receptor (nuclear receptor superfamily)
TR Thyroid hormone receptor (nuclear receptor superfamily)
C/EBP CAAT/enhancer binding protein
E2F E2 factor (named for the adenovirus E2 gene)
p53 p53 (tumor suppressor protein)
Myc Product of the c-myc protooncogene (dimerizes with Max)
 Zinc finger transcription factors
A C2H2 zinc finger

Cys His Cys His

Zn
Zn
Cys His His
Cys

• each “zinc finger” consists of antiparallel b-sheets and an a-helix

• there are approximately 30 amino acid residues per finger domain
• a zinc atom is bound to two cysteine and two histidine residues (in C 2H2)
• zinc finger proteins can have from 2 to over 30 zinc finger domains
• zinc fingers of transcription factors bind to the major groove of DNA
• examples of zinc finger transcription factors include Sp1 and the
steroid hormone receptors (nuclear receptor superfamily)
• some zinc fingers do not contain histidine (e.g., C 4 and C5 zinc fingers)
The estrogen receptor
DNA binding domain

hormone binding, dimerization

N transactivation C 4 + C5 and transactivation
C

A C4 + C5 zinc finger pair

Cys Cys Cys Cys

Zn Zn
Cys Cys Cys Cys

Cys
Model for binding of steroid receptor dimer to DNA

one steroid receptor

monomer
(with two zinc fingers)

the other steroid receptor

monomer
(with two zinc fingers)
 Binding of the estrogen receptor (ER) to DNA

• two subunits of an estrogen

receptor dimer are shown
bound to DNA
A G G T C A N N N T G A C C

T C C A G T N N N A C T G G

• each subunit has one of its

two zinc fingers nestled into
the major groove of the DNA

• the amino acid side chains of

the zinc fingers recognize the
DNA bases in dsDNA in a
sequence-specific fashion

5’-AGGTCANNNTGACCT-3’
:::::::::::::::
T

3’-TCCAGTNNNACTGGA-5’
Estrogen response element (ERE)
Steroid hormone action in target cells
mifepristone (RU486) is a
progesterone receptor antagonist
 Mutations affecting promoters
The factor IX gene
• located on the X chromosome
• transcribed region >32,700 bp, with 8 exons
The factor IX gene promoter
• there are overlapping binding sites for AR and HNF4

-36 -27 -22 -15

AR HNF4
• AR = androgen receptor
• zinc finger nuclear receptor superfamily transcription factor
• binds androgen
• androgen levels increase at puberty
• HNF4 = hepatocyte nuclear factor-4
• zinc finger nuclear receptor superfamily transcription factor
• ligand unknown - therefore an “orphan” receptor
• HNF4 is expressed early in development and in adult liver
 • mutation at -20 results in
Hemophilia B Leyden in which
the hemophilia improves at puberty
when levels of androgen increase

-36 -27 -22 -15

AR HNF4

• mutation at -26 results in

Hemophilia B Brandenburg
in which factor IX levels remain low even after puberty
 RNA Processing

a). Steps in mRNA processing

i). Capping
ii). Cleavage and polyadenylation
iii). Splicing
b). Chemistry of mRNA splicing
c). Spliceosome assembly and splice site recognition
i). Donor and acceptor splice sites
ii). Small nuclear RNAs
d). Mutations that disrupt splicing
e). Alternative splicing
 Learning Objectives for Lecture 6:

• Know the major steps in processing eukaryotic mRNA

• Understand how the two transesterification reactions
remove an intron transcript and ligate the exon
transcripts
• Understand the nature of the donor and acceptor splice
sites
• Understand what a spliceosome is and how splicing
requires small nuclear RNAs
• Understand how splice sites are selected
• Understand how mutations in splice sites affect mRNA
production
• Understand how different patterns of alternative splicing
can give rise to a diversity of mRNAs and proteins
Learning Objectives :

• Know the major steps in processing eukaryotic mRNA

• Understand how the two transesterification reactions remove an intron
transcript and ligate the exon transcripts
• Understand the nature of the donor and acceptor splice sites
• Understand what a spliceosome is and how splicing requires small nuclear
RNAs
• Understand how splice sites are selected
• Understand how mutations in splice sites affect mRNA production
• Understand how different patterns of alternative splicing can give rise to a
diversity of mRNAs and proteins
Steps in mRNA processing (hnRNA is the precursor of mRNA)
• capping (occurs co-transcriptionally)
• cleavage and polyadenylation (forms the 3’ end)
• splicing (occurs in the nucleus prior to transport)

exon 1 intron 1 exon 2

Transcription of pre-mRNA and capping at the 5’ end

cap

Cleavage of the 3’ end and polyadenylation

cap
cap poly(A)

Splicing to remove intron sequences

cap poly(A)

Transport of mature mRNA to the cytoplasm

Capping occurs co-transcriptionally shortly after initiation
• guanylyltransferase (nuclear) transfers G residue to 5’ end
• methyltransferases (nuclear and cytoplasmic) add methyl
groups to 5’ terminal G and at two 2’ ribose positions on
the next two nucleotides
pppNpN

mGpppNmpNm

capping involves formation of a 5’- 5’ triphosphate bond

• cap function
• protects 5’ end of mRNA (increases mRNA stability)
Polyadenylation
• cleavage of the primary transcript occurs approximately
10-30 nucleotides 3’-ward of the AAUAAA consensus site
• polyadenylation catalyzed by poly(A) polymerase
• approximately 200 adenylate residues are added
cleavage
AAUAAA
mGpppNmpNm

AAUAAA A A
A
polyadenylation
mGpppNmpNm A
A
A
3’

• poly(A) is associated with poly(A) binding protein (PBP)

• function of poly(A) tail is to stabilize mRNA
Chemistry of mRNA splicing
• two cleavage-ligation reactions
• transesterification reactions - exchange of one
phosphodiester bond for another - not catalyzed by
traditional enzymes
• branch site adenosine forms 2’, 5’ phosphodiester bond
with guanosine at 5’ end of intron

intron 1

Pre-mRNA
2’OH-A branch site adenosine

exon 1 exon 2
5’ G-p-G-U
- A-G-p-G 3’

First clevage-ligation (transesterification) reaction

 • ligation of exons releases lariat RNA (intron)

intron 1 Splicing
intermediate
U-G-5’-p-2’-A
A

exon 1 exon 2
5’ G-OH
O 3’ A-G-p-G
A - 3’

Second clevage-ligation reaction

intron 1
Lariat

U-G-5’-p-2’-A
A

3’ G-A
Spliced mRNA
exon 1 exon 2
5’ G-p-G 3’
Recognition of splice sites
• invariant GU and AG dinucleotides at intron ends
• donor (upstream) and acceptor (downstream) splice sites
are within conserved consensus sequences

donor (5’) splice site branch site acceptor (3’) splice site

G/GUAAGU..................…A.......…YYYYYNYAG/G

U1 U2

• small nuclear RNA (snRNA) U1 recognizes the

donor splice site sequence (base-pairing interaction)
• U2 snRNA binds to the branch site (base-pairing interaction)

Y= U or C for pyrimidine; N= any nucleotide

Spliceosome - assembly of the splicing apparatus
• snRNAs are associated with proteins (snRNPs or “snurps”)
• splicing snRNAs - U1, U2, U4, U5, U6
• antibodies to snRNPs are seen in the autoimmune
disease systemic lupus erythematosus (SLE)

= hnRNP proteins
Spliceosome assembly
intron 1
Step 1: binding of U1
and U2 snRNPs
U2
2’OH-A

exon 1 exon 2

5’ U1
G-p-G-U
- A-G-p-G 3’
 intron 1 Step 2: binding of U4, U5, U6

U2 2’OH-A

exon 1
U4 U6 exon 2

5’
U5
G-p-G-U
- A-G-p-G 3’

U1

Step 3: U1 is released,
intron 1
then U4 is released

2’OH-A

U2
exon 1
U6 exon 2

5’ G-p-G-U
- U5 A-G-p-G 3’
Step 4: U6 binds the 5’ splice site and
the two splicing reactions occur,
catalyzed by U2 and U6 snRNPs

intron 1
2’OH-A

U6 U2
U-G-5’-p-2’-A
A

mRNA 3’ G-A U5
5’ G-p-G 3’
Frequency of bases in each position of the splice sites

Donor sequences

exon intron
%A 30 40 64 9 0 0 62 68 9 17 39 24
%U 20 7 13 12 0 100 6 12 5 63 22 26
%C 30 43 12 6 0 0 2 9 2 12 21 29
%G 19 9 12 73 100 0 29 12 84 9 18 20
A G G U A A G U

Acceptor sequences

intron exon
%A 15 10 10 15 6 15 11 19 12 3 10 25 4 100 0 22 17
%U 51 44 50 53 60 49 49 45 45 57 58 29 31 0 0 8 37
%C 19 25 31 21 24 30 33 28 36 36 28 22 65 0 0 18 22
%G 15 21 10 10 10 6 7 9 7 7 5 24 1 0 100 52 25
Y Y Y Y Y Y Y Y Y Y Y N Y A G G
Polypyrimidine track (Y = U or C; N = any nucleotide)
Mutations that disrupt splicing
• bo-thalassemia - no b-chain synthesis
• b+-thalassemia - some b-chain synthesis

Normal splice pattern:

Exon 1 Exon 2 Exon 3

Intron 1 Intron 2

Donor site: /GU Acceptor site: AG/

Intron 2 acceptor site bo mutation: no use of mutant site; use of cryptic splice site in intron 2
Translation of the retained
portion of intron 2 results
Exon 1 Exon 2 in premature termination
Intron 1 of translation due to a stop
codon within the intron, 15
codons from
Intron 2 cryptic acceptor site: UUUCUUUCAG/G the cryptic splice site

mutant site: GG/

Intron 1 b+ mutation creates a new acceptor splice site: use of both sites

Exon 1 Exon 2 Exon 3

Intron 2

Donor site: /GU AG/: Normal acceptor site (used 10% of the time in b+ mutant)

CCUAUUAG/U: b+ mutant site (used 90%of the time)

CCUAUUGG U: Normal intron sequence (never used because it does not conform to a splice site)

Translation of the retained portion of intron 1 results in termination at a stop codon in intron 1

Exon 1 b+ (Hb E) mutation creates a new donor splice site: use of both sites

Exon 2 Exon 3
Intron 2

/GU: Normal donor site (used 60% of the time when exon 1 site is mutated)

GGUG/GUAAGGCC: b+ mutant site (used 40%of the time)

GGUG GUGAGGCC: Normal sequence (never used because it does not conform to a splice site)

The GAG glutamate codon is mutated to an AAG lysine codon in Hb E

The incorrect splicing results in a frameshift and translation terminates at a stop codon in exon 2
Patterns of alternative exon usage
• one gene can produce several (or numerous) different
but related protein species (isoforms)

Cassette

Mutually exclusive

Internal acceptor site

Alternative promoters
The Troponin T (muscle protein) pre-mRNA
is alternatively spliced to give rise to
64 different isoforms of the protein

Constitutively spliced exons (exons 1-3, 9-15, and 18)

Mutually exclusive exons (exons 16 and 17)

Alternatively spliced exons (exons 4-8)

Exons 4-8 are spliced in every possible way

giving rise to 32 different possibilities

Exons 16 and 17, which are mutually exclusive,

double the possibilities; hence 64 isoforms