Transcription and Splicing machinery
DNA  primary mRNA  mature mRNA
Transcription + Processing
1
Prokaryotic and Eukaryotic RNA Polymerases are similar in shape
Sigma (σ) subunit missing
-> Different number of subunits
2
Recognizes the promoter site (-10 box + -35 box)
3
RNA polymerase mechanism
-> Similar to DNA polymerase
-> 3’-hydroxyl group of RNA chain
attacks the a-phosphoryl group of the
incoming NTP
-> Transition state stabilized by Mg2+
4
Transcription
AFM image of short DNA
fragment with RNA polymerase
molecule bound to transcription
recognition site. 238nm scan size.
Courtesy of Bustamante Lab,
Chemistry Department, University
of Oregon, Eugene OR
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6
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Prokaryotic promoter sites
-35
-10
+1
5’-----TTGACA--------------TATAAT---------start site----3’
σ subunit
8
Prokaryotic promoter sites
σ subunit interacts with
-10 box and -35 box
Alternative E. coli promoters
Stanard Promoter
-> σ70
Heat shock promoter
-> σ32
N-starvation promoter -> σ54
10
Footprinting
11
DNA unwinding prior to Initiation of Transcription
-> Transition from closed to open complex
-> Unwinding done by RNA polymerase
1 RNA polymerase molecule -> 17bp segment ->
1.6 turns on B-DNA
12
Negative supercoiled DNA favors the transcription
-> neg. supercoiling facilitates unwinding
-> introduction of neg. supercoiling -> increases rate of transcription
-> Exception -> promoter of TopoII -> neg. Supercoiling -> decreases
rate of transcription
13
Transcription bubble
First Nucleotide is pppG or pppA -> Transcription start
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RNA-DNA hybrid separation
RNA polymerase
forces the
separation of the
RNA-DNA hybrid
15
Transcription Termination
Rho independent termination
-> RNA polymerase pauses after
production of hairpin
-> RNA-DNA hybrid of hairpin is unstable
=> RNA falls off
Termination by Rho protein
Rho interacts with RNA polymerase ->
breaks the RNA-DNA hybrid helix ->
functions as a helicase
16
Primary transcript of rRNA is modified
Modification: 1. Cleavage of primary transcript by Ribonuclease III
2. Modification of bases (Prokaryotes: methylation)
and ribose (Eukaryotes: methylation)
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tRNA transcript is also modified
Modification: 1. Cleavage of primary transcript by Ribonuclease III
2. Addition of nucleotides at 3’ end (CCA)
3. Unusual bases
18
tRNA transcript processing
Modification: 1. Cleavage of primary transcript by Ribonuclease III
2. Addition of nucleotides at 3’ end (CCA)
3. Unusual bases
19
Antibiotic Inhibitors of Transcription
Rifampicin: - derivate of rifamycin (Streptomyces)
- inhibits initiation of RNA synthesis (binds to RNA polymerase -> in pocket
where RNA-DNA hybrid is formed)
Actinomycin D: - polypeptide-containing (Streptomyces)
- binds tightly (intercalates) to ds-DNA (cannot be template for RNA
synthesis)
- its ability to inhibit growth of rapid dividing cells makes it a effective
agent in cancer treatment
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Transcription and Translation in Prokaryotes and Eukaryotes
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α-Amanitin:
produced by mushroom (Amanita phalloides)
-> cyclic peptide of 8 amino acids
-> binds tightly to RNA polymerase II
-> blocks elongation of RNA synthesis
-> deadly doses (LD50 is 0.1 mg/kg)
22
Different Eukaryotic RNA Polymerase promoters
Inr -> Initiator element
(found at transcription
start)
DPE -> downstream core
promoter element
Eukaryotic promoter elements
(RNA polymerase II promoter)
-> -40 and -150
Normally between -30 and -100
Often paired with Inr -> -3 and -5
CAAT boxes and GC boxes can even be on
noncoding strand active
DPE -> +28 and +32
24
Eukaryotic Transcription Initiation
TappingMode AFM image of an individual
human transcription factor 2: DNA
complex. Clearly resolved are the
protein:protein interactions of two
transcription factor proteins which
facilitate the looping of the DNA, allowing
two distal DNA sites to be combined.
AFM provided the investigators' improved
resolution of the looped DNA complexes
compared to electron microscopy of
rotary shadowed samples. 252 nm scan.
Image courtesy of Bustamante Lab,
Institute of Molecular Biology, University
of Oregon, Eugene.
25
Eukaryotic Transcription Initiation
Basal
transcription
apparatus
(-> carboxylterminal domain)
TATA-box binding protein (TBP
is a component of TFIID)
recognizes the TATA box and
forms complex with DNA
CTD plays a role in transcrition regulation ->
binds to mediator
Phosphorylation of CTD by TFIIH -> elongation
of transcription
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Eukaryotic Transcription Initiation Complex
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Regulation of Transcription
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Packaging of Eukaryotic chromosomal DNA
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Transcription Initiation
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Gene “Off”
Gene “On”
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32
Eukaryotic transcription products (from RNA polymerase II) are processed
triphosphate
7-methylguanylate
end
Capping 5’ end
Polyadenylation of 3’ end
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RNA editing
34
Splicing
Anemia: defect synthesis of
hemoglobin
Mutations affecting splice sites cause
Creates a new splice site
around 15% of all genetic diseases
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Small nuclear RNAs in spliceosomes catalyse the splicing
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Spliceosome assembly
The catalytic center of the spliceosome
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Alternative splicing
39
Self-splicing
A rRNA precursor of Tetrahymena (protozoan) splices
itself in the presence of guanosine (G) as co-factor
The L19 RNA is a intron that is catalytical active
This TappingMode scan of the protozoan, Tetrahymena,
shows its cilia-covered body and mouth structures. The
sample was dried onto a glass slide and scanned; no other
preparation was required. 50 micron scan courtesy C.
Mosher and E. Henderson, BioForce Laboratory and Iowa
State University.
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Self-splicing mechanism
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Ribosomal Factory
Protein
mRNA
Translation
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Translation:
mRNA -> Protein
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Peptide bond formation in Ribosomes
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Linkage of Amino Acids to tRNA
2nd step
1st step
Linkages either 2’ or 3’
1st step: activation of AA by adenylation (Aminoacyl-AMP)
2nd step: linkage of AA to tRNA
48
Aminoacyl-tRNA synthetases couple Amino acids to tRNA
Synthetases are highly specific for the amino acid (error rate
1 in 105)
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50
Proofreading of Aminoacyl-tRNA Synthetases
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Synthetases recognize the anticodon loops and acceptor stems of tRNA
Threonyl-tRNA synthetase complex
Class II synthetase
Glutaminyl-tRNA synthetase complex
Class I synthetase
52
Classification of Aminoacyl-tRNA synthetases
Synthetases recognize different faces of the tRNA molecule:
1.
Class I acylates the 2’ OH group of the terminal adenosine of tRNA
2.
Class II acylates the 3’ OH group of the terminal adenosine of tRNA
3.
They bind ATP in different conformations
4.
Most class I are monomeric, most class II are dimers
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54
Ribosomes are Ribonucleoproteins
50S
70S
30S
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Ribosomal RNAs (5S, 16S, 23S rRNA)
16S rRNA
tertiary structure
secondary structure
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Ribosomal Protein L19 of the 50S subunit
Fits through some of the cavities within the 23S RNA
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Protein synthesis in E. coli
Polysomes: Transcription and
Translation happens at the same time
Direction of Transcription: 5’->3’
Direction of Translation: 5’->3’
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Translational Initiation sites – Ribosome binding sites
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Bacterial Protein synthesis is
initiated by Formylmethionyl
tRNA -> fMet
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tRNA binding sites on Ribosomes
A for aminoacyl -> tRNA enters Ribosomes
P for peptidyl -> tRNA passed on - peptide bonds are closed
E for exit -> tRNA exits Ribosomes
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Polypeptide chain escape path
Polypeptide synthesis tunnel
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Peptide bond formation
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Some tRNAs recognize more than one codon -> wobble base
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Elongation factor Tu
EF-Tu delivers aminoacyltRNA to Ribosomes
Elongation factor G
EF-G mediates translocation
within the Ribosome
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Translocation mechanism
EF-G (in GTP form) binds to EF-Tu site -> stimulates GTP hydrolysis
Conformational change of EF-G -> driving EF-G into A site
Causes translocation of tRNA and mRNA
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Diphtheria Toxin blocks Protein Synthesis by Inhibition of Translocation
Disease: Diphtheria
Cause: Toxin from
Corynebacterium diphtheriae
Toxin catalysis transfer of
ADP-ribose to diphthalamide ( a
modified AA in EF 2 –
translocase)
68
Protein Synthesis Termination by Release Factors
69
Differences between Eukaryotic and Prokaryotic Protein Synthesis
Difference -> Translocation Initiation
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Protein Interaction cirularize mRNA