TRANSCRIPTION LECTURES, FALL 2010

NABIL BASHIR

RNA Structure and Transcription- Prokaryotes

a). Chemistry of RNAi). Bases found in RNAii). Ribose sugariii). RNA polynucleotide chainiv). Secondary and tertiary structure

b). Characteristics of prokaryotic RNAi). Classes of prokaryotic RNAii). Structure of prokaryotic messenger RNA

c). Transcription initiation in prokaryotesi). Transcriptionii). Promoter structureiii). Prokaryotic RNA polymerase structureiv). 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)

uracil-adenine base pairthymine-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

Tertiary structure

Secondary structure

• 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

5’

3’

PuPuPuPuPuPuPuPu AUGShine-Dalgarno sequence initiation

The Shine-Dalgarno (SD) sequence base-pairs with a pyrimidine-rich sequence in 16S rRNA to facilitate the initiation of protein synthesis

Classes of prokaryotic RNA

AAUtermination

translated region

Transcription

RNA polymerase

closed promoter complex

open promoter complex

initiation

elongation

termination

RNA product

Promoter structure in prokaryotes

5’ PuPuPuPuPuPuPuPu AUG

Promoter

+1 +20-7-12-31-36

5’mRNA

mRNA

TTGACAAACTGT

-30 region

TATAATATATTA

-10 region

84 79 53 45%82T T G

64AC A

79T

44T

96%T

95A

59A

51A

consensus sequences

-30 -10

transcription start site

Pribnow box

+1[ ]

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’ + sholoenzyme 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

RNA polymerase holoenzyme (+ s factor)

• closed promoter complex (moderately stable)• the sigma subunit binds to the -10 region

• 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

• elongation takes place with the core RNA polymerase

• open promoter complex (highly stable)• the holoenzyme has very high affinity for promoter regions because of sigma factor

s• sigma can re-bind other core enzymes The sigma cycle

s

s

Mechanism of RNA synthesis

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

A = T

U = A

A = T

U = A

RNA RNA

Eukaryotic Transcriptional Regulation

a). Characteristics of eukaryotic RNA and their polymerasesi). Classes of cellular RNAii). RNA polymerases I, II, and III

b). Transcription of messenger RNA in eukaryotesi). Structure of eukaryotic messenger RNAii). Complexity of mRNA populations in the celliii). Promoters and transcription elementsiv). Transcription factors

General transcription factorsBasic region-leucine zipper proteinsZinc 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 RNA _____________________________________________________________________________________________

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

7mGpppNCap

5’5’ untranslated region

AUGinitiation

translated region

(A)~200

poly(A) tail

3’ untranslated region

UGAtermination

3’AAUAAApolyadenylation signal

• 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 9 108,000intermediate 300 700 210,000low (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

1determined in RNA-DNA hybridization experiments analogous to Cot curves2mouse 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 unit

exon exonpromoter

PTE

transcription element

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

+1

Transcription and promoter elements for RNA polymerase II

transcription unit

exon exonpromoter

PTE

exon exon

transcription element

promoter complex

PTE

exon exonP TE

exon exonP TE

transcription element

TE

LCR

TE

P

PTE

locus control region

• a single locus control region (LCR) may control two or more transcription units in a cell-specific fashion

gene A

gene B

The locus control region is a specialized transcription element

Sequence elements within a typical eukaryotic gene1

GC TATACAAT GC

-25-50-80-95-130

1 based on the thymidine kinase gene octamertranscription element

promoter

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)

+1

ATTTGCAT

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

1TFIIH is also involved in phosphorylation of RNA polymerase II, DNA repair

(Cockayne syndrome mutations), and cell cycle regulation

+1

TBP

TFIID

A

B

E F

H

J

-25

TAFs

Binding of the general transcription factors

• TFIID (a multisubunit protein) binds to the TATA boxto 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

RNA pol II

TBP

TFIID

A

B

E F

H

J

• 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 RNA polymerase II

+1

TBP

TFIID

A

B

E F

H

J

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”

Binding of specialized TFs

+1

TBP

TFIIDB

E F

H

J

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

Formation of a stable preinitiation complex

+1

TBP

TFIIDB

E F

H

J RNA pol II

initiation

• 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

Initiation of transcription and promoter clearance

P

PP

CTD

Transcription factors (partial list)

Factor Full name or function

CREB Cyclic AMP response element binding proteinCTF 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 kBHSTF Heat shock transcription factorMTF Metal transcription factorUSF Upstream factorATF Activating transcription factorHNF4 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 proteinE2F 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, tumorpromoters, neuronal stimulation, and cellular stress

• Four leucines ( ) are present at every seventh position in the amphipathic a-helix

The Fos and Jun proteins

Fos Jun

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

Leucine zipper (dimerization domain)

Helical wheel analysis of a leucine half-zipper

Leucine at every seventh position

Amphipathic alpha helix

Dimerization of the AP1 transcription factor

N

C

N

C

• The leucine zippers interact via their hydrophobic faces forming coiled coils that cause the two proteins to dimerize

• Dimerization via the leucine 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

DNA binding domains

dimerized leucinezippers

Gcn4 (Basic Region, Leucine Zipper) Complex With Ap-1 DNA

Structures generated using RasWin Molecular GraphicsWindows Version 2.6 and PDB ID# 1YSA

DNA binding

Leucine zipper

+1

TBP

TFIID

A

B

E F

H

J

RNA pol II

Binding of AP1 to DNA transactivates transcription

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

TGACTCAACTGAGT

+1

TBP

TFIID

A

B

E F

H

J

RNA pol II

Binding of AP1 to DNA transactivates transcription

• 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)

P

TGACTCAACTGAGT

Fos Jun

Transcription factors (partial list)

Factor Full name or function

CREB Cyclic AMP response element binding proteinCTF 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 kBHSTF Heat shock transcription factorMTF Metal transcription factorUSF Upstream factorATF Activating transcription factorHNF4 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 proteinE2F 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

His

HisCys

Zn

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 C2H2)• 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., C4 and C5 zinc fingers)

ZnCys

Cys

His

His

A C2H2 zinc finger

The estrogen receptor

A C4 + C5 zinc finger pair

ZnCys

Cys

Cys

Cys

Cys

ZnCys

Cys

Cys

Cys

C4 + C5transactivation

hormone binding, dimerization and transactivation

DNA binding domain

N C

Model for binding of steroid receptor dimer to DNA

one steroid receptormonomer

(with two zinc fingers)

the other steroid receptormonomer

(with two zinc fingers)

Binding of the estrogen receptor (ER) to DNA

• two subunits of an estrogen receptor dimer are shown bound to DNA

• 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’ :::::::::::::::3’-TCCAGTNNNACTGGA-5’

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

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

A

Estrogen response element (ERE)

Steroid hormone action in target cells

mifepristone (RU486) is aprogesterone receptor antagonist

The factor IX gene promoter• there are overlapping binding sites for AR and 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

Mutations affecting promoters

The factor IX gene• located on the X chromosome• transcribed region >32,700 bp, with 8 exons

-27 -15-36 -22

HNF4AR

• mutation at -20 results in Hemophilia B Leyden in which the hemophilia improves at puberty when levels of androgen increase

-27 -15-36 -22

HNF4AR

• mutation at -26 results inHemophilia B Brandenburgin which factor IX levels remain low even after puberty

RNA Processing

a). Steps in mRNA processingi). Cappingii). Cleavage and polyadenylationiii). Splicing

b). Chemistry of mRNA splicingc). Spliceosome assembly and splice site recognition

i). Donor and acceptor splice sitesii). Small nuclear RNAs

d). Mutations that disrupt splicinge). 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

cap

cap

cap poly(A)

cap poly(A)

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

Cleavage of the 3’ end and polyadenylation

Splicing to remove intron sequences

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 onthe next two nucleotides

capping involves formation of a 5’- 5’ triphosphate bond• cap function

• protects 5’ end of mRNA (increases mRNA stability)• required for initiation of protein synthesis

pppNpN

mGpppNmpNm

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

• poly(A) is associated with poly(A) binding protein (PBP)• function of poly(A) tail is to stabilize mRNA

mGpppNmpNmAAUAAA

mGpppNmpNmAAUAAA AA

A

A

AA

3’

cleavage

polyadenylation

Chemistry of mRNA splicing• two cleavage-ligation reactions• transesterification reactions - exchange of one

phosphodiester bond for another - not catalyzed bytraditional enzymes

• branch site adenosine forms 2’, 5’ phosphodiester bondwith guanosine at 5’ end of intron

G-p-G-U A-G-p-G

2’OH-A

-5’ 3’

intron 1

exon 1 exon 2

Pre-mRNA

First clevage-ligation (transesterification) reaction

branch site adenosine

G-OH 3’ A-G-p-G

U-G-5’-p-2’-A

5’ 3’A

A

O -

G-p-G5’ 3’

U-G-5’-p-2’-AA

3’ G-A

Splicingintermediate

Lariat

exon 1

exon 1

exon 2

exon 2

intron 1

intron 1

Second clevage-ligation reaction

Spliced mRNA

• ligation of exons releases lariat RNA (intron)

Recognition of splice sites• invariant GU and AG dinucleotides at intron ends• donor (upstream) and acceptor (downstream) splice sites

are within conserved consensus sequences

• small nuclear RNA (snRNA) U1 recognizes thedonor 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

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

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

U1 U2

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)

G-p-G-U A-G-p-G

2’OH-A

-5’ 3’

intron 1

exon 1 exon 2

Spliceosome assembly

Step 1: binding of U1and U2 snRNPs

U1

= hnRNP proteins

U2

G-p-G-U A-G-p-G

2’OH-A

-5’ 3’

intron 1

exon 1 exon 2

Step 2: binding of U4, U5, U6

U1

U5

U2U4 U6

G-p-G-U A-G-p-G

2’OH-A

-5’ 3’

intron 1

exon 1 exon 2

Step 3: U1 is released,then U4 is released

U5

U2U6

G-p-G5’ 3’

U-G-5’-p-2’-AA

3’ G-A

intron 1

mRNA

2’OH-A

U5

U2U6

Step 4: U6 binds the 5’ splice site andthe two splicing reactions occur,catalyzed by U2 and U6 snRNPs

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 3Intron 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

Exon 1 Exon 2Intron 1

mutant site: GG/

Intron 2 cryptic acceptor site: UUUCUUUCAG/G

Translation of the retained portion of intron 2 results in premature termination of translation due to a stop codon within the intron, 15 codons fromthe cryptic splice site

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

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 Exon 2 Exon 3Intron 2

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

Exon 2 Exon 3Intron 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-mRNAis alternatively spliced to give rise to64 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 waygiving rise to 32 different possibilities

Exons 16 and 17, which are mutually exclusive,double the possibilities; hence 64 isoforms