Transcription 11/3/05
Stable RNA
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rRNA -Structural component of ribosomes
tRNA-Adaptors, carry aa to ribosome
Synthesis
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Promoter and terminator
Post-transcriptional modification (RNA
processing)
Evidence
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Both have 5’ monophospates
Both much smaller than primary transcript
tRNA has unusual bases. EX pseudouridine
tRNA and
rRNA
Processing
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Both are
excised from
large primary
transcripts
1º transcript
may contain
several tRNA
molecules, tRNA
and rRNA
rRNAs simply
excised from
larger transcript
tRNAs modified
extensively
5. Base
modifications
Examples of Covalent Modification of Nucleotides in tRNA
CH3
H
H
CH3
N
N
6
N
H
N
N
6
N
N
N
C
C
CH3
CH2
O
N
N
N6-Methyladenylate
N6-Isopentenyladenylate
(m6A)
(i6A)
O
H
H
H
H
NH
HN 3
4
N
O
Dihydrouridylate
(D)
2
1
5
C
H2C
NH
O
N
O
Uridylate
5-oxyacetic acid
(cmo5U)
Pseudouridylate (Ψ)
(ribose at C-5)
N
N
3
N
CH3
6
CH2
5
NH
NH
5
NH2
7-Methylguanylate
(m7G)
O
O
N
N
O
3-Methylcytidylate
(m3C)
O
NH2
H3C
N
N
O
C
NH
7
Inosinate
(I)
HO
O
N
NH
N
N
O
CH3
O
5-Methylcytidylate
(m5C)
Base
O
H
H
O
H
O CH3
H
1'
2'
2’-O-Methylated nucleotide
(Nm)
Modifications are
shown in blue.
Eukaryotic Transcription
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Regulation very complex
Three different pols
Distinguished by -amanitin sensitivity
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Pol I—rRNA, least sensitive
Pol II– mRNA, most sensitive
Pol III– tRNA and 5R RNA moderately
sensitive
Each polymerase recognizes a distinct
promoter
Eukaryotic RNA Polymerases
RNA Pol. Location Products
I
Nucleolus Large rRNAs
(28S, 18S,
5.8S)
-Amanitin Promoter
Sensitivity
Insensitive
II
Nucleus
Pre-mRNA,
some snRNAs
Highly
sensitive
III
Nucleus
tRNA, small
rRNA (5S),
snRNA
Intermediate
sensitivity
bipartite
promoter
Upstream
Internal
promoter and
terminator
Eukaryotic Polymerase I Promoters
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RNA Polymerase I
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Transcribes rRNA
Sequence not well conserved
Two elements
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Core element- surrounds the transcription
start site (-45 to + 20)
Upstream control element- between -156 and
-107 upstream
Spacing affects strength of transcription
Eukaryotic Polymerase II Promoters
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Much more complicated
Two parts
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Core promoter
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Core promoter
Upstream element
TATA box at ~-30 bases
Initiator—on the transcription start site
Downstream element-further downstream
Many natural promoters lack recognizable
versions of one or more of these sequences
TATA-less Promoters
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Some genes transcribed by RNA pol II lack the
TATA box
Two types:
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Housekeeping genes ( expressed constitutively). EX
Nucleotide synthesis genes
Developmentally regulated genes. EX Homeotic genes
that control fruit fly development.
Specialized (luxury) genes that encode cell-type
specific proteins usually have a TATA-box
mRNA Processing in Eukaryotes
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Primary transcript much larger than
finished product
Precursor and partially processed RNA
called heterogeneous nuclear RNA
(hnRNA)
Processing occurs in nucleus
Capping mRNA
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5’ cap is a reversed
guanosine residue so
there is a 5’-5’ linkage
between the cap and
the first sugar in the
mRNA.
Guanosine cap is
methylated.
First and second
nucleosides in mRNA
may be methylated
BACK
Polyadenylation
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Polyadenylation occurs on the 3’ end of
virtually all eukaryotic mRNAs.
Occurs after capping
Catalyzed by polyadenylate polymerase
Polyadenylation associated with mRNA
half-life
Histones not polyadenylated
Introns and Exons
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Introns--Untranslated
intervening
sequences in mRNA
Exons– Translated
sequences
Process-RNA splicing
Heterogeneous
nuclear RNA
(hnRNA)-Transcript
before splicing is
complete
Splicing Overview
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Occurs in the nucleus
hnRNAs complexed with specific proteins,
form a ribonucleoprotein particle (RNP)
Primary transcripts assembled into hnRNP
Splicing occurs on spliceosomes consist of
Small nuclear ribonucleoproteins (SnRNPs)
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components of spliceosomes
Contain small nuclear RNA (snRNA)
Many types of snRNA with different functions
in the splicing process
Splice Site Recognition
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Introns contain invariant 5’-GU and 3’-AG
sequences at their borders (GU-AG Rule)
Internal intron sequences are highly variable even
between closely related homologous genes.
Alternative splicing allows different proteins from
a single original transcript
Simplified Splicing Mechanism
RNA pol III
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Precursors to tRNAs,5SrRNA, other small RNAs
Promoter Type I
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Lies completely within the transcribed region
5SrRNA promoter split into 3 parts
tRNA promoters split into two parts
Polymerase II-like promoters
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EX. snRNA
Lack internal promoter
Resembles pol II promoter in both sequence and
position
DNAse
Footprinting
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Protected region
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Use: promoter
ID
End Label
template strand
Add DNA
binding protein
Digest with
DNAse I
Remove protein
Separate on gel