start of transcription 5’ pppa upstream 3’...cpapgpgptpgpcpapgptpgpcptp...5’ downstream dna...
Post on 20-Dec-2015
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TRANSCRIPT
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.