Start of Transcription 5’ pppA Upstream 3’...CpApGpGpTpGpCpApGpTpGpCpTp...5’ Downstream DNA template strand (other strand not shown) OH 3' riboATP Point.

Start of Transcription

Start of Transcription

5’ pppA Upstream 3’...CpApGpGpTpGpCpApGpTpGpCpTp...5’ Downstream

DNA template strand (other strand not shown)

OH 3'riboATP

Point where transcription will start

5’…GpTpCpCpApCpGpTpCpApCpGpAp...3’ Upstream 3’...CpApGpGpTpGpCpApGpTpGpCpTp...5’ Downstream

DNA template strand

Transcriptional initiation complex

Elongation of a transcript

Start of Transcription

pppA Upstream ...CpApGpGpTpGpCpApGpTpGpCpTp... Downstream

DNA template strand (other strand not shown)

pppC-OH incoming nucleoside (CTP) triphosphate

OH 3'RNA

Incorporated nucleotide

pppApCpGpUpC...CpApGpGpTpGpCpApGpTpGpCpTp....

OH 3’ H2O ppi 2pi

pyrophosphate phosphate

+

Hydrolysis of pyrophosphate (by pyrophosphatase) is an important driving force.

RNA polymerase

RNA polymerase I

• Synthesis of– 18S rRNA– 5.8S rRNA– 28S rRNA

(5S rRNA is synthesized by polIII)

rRNA genes are located on the “stalk” regions of chromosomes 13, 14. 15. 21 & 22

rRNA genes are located in tandem arrays

rRNAgene rRNAgene rRNAgene

DNA

RNAsRNA pol I

RNA polymerase 1

RNA processing enzymes

rRNA Processing(occurs in the nucleolar regions of the nucleus)

18S 5.8S 28S

5’ppp

3’ OH

rRNA gene (DNA)

Precursor RNA

Mature RNAs

Introducing the concept of RNA processing

RNA polymerase II

• Synthesis of mRNA (and some small RNAs)

RNA polymerase III

• Synthesis of small RNAs including– 5S rRNA– tRNAs

• Located in the nucleus (not nucleolus)

Details of pol III promoters are not essential for this course

mRNA Structure

7 methyl-G cap

5’ untranslated region

Start Codon:AUG

Stop Codon: UGA UAA UAG

Poly A signalAAUAAA

3’ untranslated region

A200

3’ poly A tail Coding region; ORF(Open Reading Frame)

Structure of the mRNA cap

NO

N

N

OON

NH2

O

N

N

N+

O

NH

O

NH2

OH OH

OCH3 P

O

OO P

O

O

O

P

O

O

PO

O

O

CH3

Continuation of the mRNA

2' OH may be methylated(the second nucleotie may also have a 2' methyl)

7-methy G Note the positive charge Triphosphate in a 5' to 5' linkage

First residue of the chain (usually an A)

Structure of the eukaryotic mRNA cap

Overview of pre-mRNA processing

Primary transcript:

RNA capping: 7mG

3’end cleavage: 7mGpolyadenlyation signal

polyadenylation: 7mG An

RNA splicing: 7mG An

(RNA splicing may precede, occur at the same time as, or follow 3’ end formation)

Transport through a nuclear pore complex to the cytoplasm

Check by nonsense mediated decay pathway

3’ end Formation

Stop Codon polyadenylation signal (e.g.AAUAAA)

3’ Processing complex

3’end cleavage

Poly A addition

Last exon

3’ untranslated region

Mutations that affect 3’ end formation

• Example: hyperprothrombinemia– Due to a G-to-A transition at position 20,210– Mutation causes an increase in the amount of

prothrombin– Associated with about a 3-fold increased risk of

myocardial infarction– Present in about 2% of the European population

but rare in non-caucasians

Mutations that affect 3’ end formation

• Example: hyperprothrombinemia– 20210 is the last nucleotide before the polyA tail

– Due to increased 3’ end formation, cells with the 20210A allele produce more prothrombin mRNA than those with the 20210G allele

– The 20210A mRNA has a longer half-life than 20210G mRNA.

Mutations that affect 3’ end formation

• Typically, if 3’end mutations have an effect, they reduce the amount of mRNA that is made.

– Example: beta-plus thalassemia

Intron 1

Overview of mRNA Splicing

Exon 1 AGGU Exon 2A AGG

Consensus sequences

A typical intron is 100 - 50,000 nucleotides long, starts with GU and ends with A(C,U)17-37AG.

This adenosine is 17-37 nucleotides from splice site.

A typical exon is 100 - 300 nucleotides long, starts with G and ends with AG.

Exon selection factor

Exon selection factor

U2 snRNPU1 snRNP

Intron 1

Overview of mRNA Splicing

Exon 1 AGGU Exon 2A AGG

Factors such as U1 and U2 snRNP identify splice sites

Exons are identified by RNA sequences within the exons that are recognized by exon selection factors.

U2U1

U2 snRNPU1 snRNP

Intron 1

Overview of mRNA Splicing

Exon 1 AGGU Exon 2A AGG

AUG

GA A

GG

HO

U2U1

U2U1

A

UGG

A AGG

HO

GG

AG

GAGOH

A

OH

O

O

O

G

O

O

O

O

P

O

O

OP

O

O

P

O O

A

2' -5' phosphodiester

Intron

Exon 2

Detail of the 2’-5’ phospodiester formed during mRNA splicing

2’-5’3’-5’

3’-5’

GG

AG

GAGOH

A2’-5’

U2U1

G

G

AG

GAG

A

GA G GG

AG

A+

degradedTo cytoplasm

OH

Alternative splicing: Isozymes of Tropomyocin

1 2 3 4 5 6 7 8 9 10 11 12

There are two alternative poly- adenylation sites. The splicing pattern determines which one is used

IntronsExons

There are two alternative translation stop codons. The splicing pattern determines which one is used

Start of transcription

Start of translation

1 2 3 4 5 6 7 8 9 10 11 12

1 4 5 6 7 8 9 122

Exons 10 and 11 are spliced out in smooth muscle.

Exon 3 is missing from smooth muscle tropomyosin mRNA.

Splicing pattern used in smooth muscle

Translation stops here.

No exon 11, so polyadenylation occurs at the end of exon 12.

Two splicing patterns are used in striated muscle

1 2 3 4 5 6 7 8 9 10 11 12

1 3 4 5 6 7 8 9 10 11

1 3 4 5 6 7 8 9 10 12

If exon 11 is not removed by splicing, poly- adenylation occurs here, and exon 12 is lost.

If splicing removes exon 11, its poly- adenylation site is removed. Polyadenylation occurs at the end of exon 12.

Translation stops here.

Translation stops here.

Exon 3, but not exon 2 is present in striated muscle tropomyosin mRNA

A.

B.