Lecture 52: Transcriptional regulation and processing
DNA
Replication
Transcription
Minireview of prokaryotic
transciption and regulation
Eukaryotic transcription,
processing and regulation
RNA
Translation
Protein
Today’s lecture: Text pp. 1058-1060, 1086-1100
Preprinted lecture notes 66-75, 97
Download from
http://biochem.med.ufl.edu/coursetemp.php?cid=44 or
http://www.mbi.ufl.edu/facilities/msg/bch4024-notes.html
Prokaryotic transcription
1)
Can be dissociated and reassociated with
RNA polymerase
a) multimeric protein complex subunits from different prokaryotes
α2ββ’σω
α2Æ Chain initiation, interacts with regulatory proteins
βÆ Chain initiation, elongation (forms phosphodiester bond)
β’Æ DNA binding
σÆ Promoter recognition and binding; loosely bound
ωÆ Unknown
b) σ subunit is important for binding to correct initiation site of a
gene (promoter)
which σ is used can be altered
based on environment
c) Core enzyme continues the polymerization reaction
2) Polymerization reaction
1) Uses ribonucleoside triphosphates
2) Requires DNA template
3) Chain growth is 5’ Æ 3’
4) Unwinds DNA in local region and displaces RNA as DNA rewinds
5) Copies only one strand of DNA
6) Disengages at specific termination sequences (symmetrical GCrich sections followed by A-rich segment) or ρ factor binds
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Prokaryotic transcription Cont’d
3)
mRNA elements
a) 5’ leader, 25-200 nucleotides with ribosome binding sequence
(Shine-Dalgarno)
b) Translation start codon: AUG (fMet)
c) Translation stop codon: UAG,UAA, UGA
4) Transcription and translation can occur simultaneously
5) Transcription can be regulated by DNA binding proteins – “helixturn-helix” motif which bind in the major grooves of the DNA
AUG
Leader
sequence
Stop codon
Transcription in Eukaryotes
1)
Several different RNA polymerases
Polymerase I: rRNA, in nucleolus
Inhibited by α-Amanitin
Require transcription factors
Polymerase II: snRNA, mRNA
Polymerase III: tRNA, 5S rRNA
Mitochondrial: similar to prokaryote RNA Pol.
Chloroplast: similar to prokaryote RNA Pol.
2) Transcriptional unit
1) Promoter
2) Start site – AUG Æ Met (not fMet)
3) Interrupted amino acid coding sequences
4) Stop site
snRNA ≡ small nuclear RNA
2
Example of Eukaryotic transcriptional unit: human β-globin
3 Exons: E1: aa 1-30
E2: aa 31-104
E3: aa 105-146
Promoter
Start
2 Introns: I1: 130 bp
I2: 900 bp
PolyA signal (AAUAAA)
RNA Polymerase II
Stop
2,3: Cap 5’ end, add poly(A) at 3’ end
m7G
AAA…A200
3,4: Remove introns, splice exons
m7G
AAA…A200
5: Transport to cytoplasm
AAA…A200
5: Translate….
Ovalalbumin gene :Exon-Intron structure
Electron micrograph of genomic DNA
and processed mRNA
Analysis
3
Transcription in Eukaryotes Cont’d
3)
5’ caps
a) Enzymatic addition by capping enzyme guanylyl transferase (+ GTP)
b)
The 5’ triphosphate on the mRNA is hydrolyzed to a diphosphate
and GMP is added in a 5’ to 5’ condensation
c)
Methylation of cap on terminal guanosine and on ribose
1) Protects mRNA from exonucleases
2) Aids in binding to ribosomes (via cap binding protein, an IF)
3) Splicing
5’ Cap
Transcription in Eukaryotes Cont’d
4)
Poly(A) addition
a) Capped mRNA cut at poly(A) signal site (AAUAAA) by a processing
endonuclease
b)
~200 A’s added by a template independent poly(A) polymerase
c)
Function of poly(A)
1) Protection
2) Transport
3) Translational enhancement
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5)
RNA Splicing (Intron removal)
Removal of (Group II) introns from mRNA:
1)
2)
Involves Spliceosomes (proteins and snRNPs)
Splicing signals and sequences
snRNP ≡ small nuclear ribonucloprotein particles
Spliceosome ≡snRNP / pre-mRNA complex
U1 RNA “recognizes” the exon-intron
junction (splice site at 5’ side of intron)
2’-OH of the bulged A attacks the 5’
splice site, forming a lariat structure
with the 5’ G of the intron. U4, U5 and
U6 join spliceosome to finish the job.
5’
5
5)
RNA Splicing (Intron removal)
1)
2)
Self-splicing RNA
(catalytic RNA or ribozymes)
Splicesosome catalyzed
splicing of nuclear RNA
“Guide sequences” in snRNAs
Transcription in Eukaryotes Cont’d
6)
Alternative splicing (immunoglobulins, etc.)
mRNA transcripts may be spliced in different
way to include or exclude certain exons.
Human
genome is not
as large as
originally
expected
α-tropomyosin: a contractile protein
6
Regulation of gene expression
occurs at three levels
1.
Transcriptional
Major regulatory control; regulates amount of primary RNA transcript
In prokaryotes: operons (Lac and Trp examples)
In eukaryotes: tomorrow (lots of factors involved)
2.
Post-transcriptional processing
A.
Alternative splicing (eukaryotes)
B.
Polyadenylation (3’), capping (5’), transport to cytoplasm (eukaryotes)
C.
Half life / turnover
3.
Translational
A.
m7G caps on mRNA (eukaryotes)
B.
Shine – Dalgarno sequences (prokaryotes)
C.
Secondary structure of mRNA
D.
Abundance of specific tRNA (minor species for low-abundance proteins)
E.
Derivatives of guanine nucleotides (“magic spots”: ppGDP, ppGTP in
metabolic control of rRNA transcription) (prokaryotes)
F.
Modification of initiation factors (ie eIF-2 phosphorylation in eukaryotes)
G.
Protein binding of mRNA
H.
Antisense RNA
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