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Transcription and Splicing machinery
DNA primary mRNA mature mRNA
Transcription + Processing
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Prokaryotic and Eukaryotic RNA Polymerases are similar in shape
-> Different number of subunitsSigma (σ) subunit missing
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Recognizes the promoter site (-10 box + -35 box)
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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+
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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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Prokaryotic promoter sites
5’-----TTGACA--------------TATAAT---------start site----3’-35 -10 +1
σ subunit
Prokaryotic promoter sites
σ subunit interacts with -10 box and -35 box
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Alternative E. coli promoters
Stanard Promoter -> σ70
Heat shock promoter -> σ32
N-starvation promoter -> σ54
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Footprinting
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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
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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
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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
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Transcription Termination
Rho independent
termination Termination by Rho protein
Rho interacts with RNA polymerase -> breaks the RNA-DNA hybrid helix -> functions as a helicase
-> RNA polymerase pauses after production of hairpin -> RNA-DNA hybrid of hairpin is unstable => RNA falls off
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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
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Modification: 1. Cleavage of primary transcript by Ribonuclease III
2. Addition of nucleotides at 3’ end (CCA)
3. Unusual bases
tRNA transcript processing
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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)
Different Eukaryotic RNA Polymerase promoters
DPE -> downstream core promoter element
Inr -> Initiator element(found at transcription start)
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Eukaryotic promoter elements (RNA polymerase II promoter)
Normally between -30 and -100
Often paired with Inr -> -3 and -5
DPE -> +28 and +32
-> -40 and -150
CAAT boxes and GC boxes can even be on noncoding strand active
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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.
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Eukaryotic Transcription Initiation
TATA-box binding protein (TBP is a component of TFIID) recognizes the TATA box and forms complex with DNA
Basal transcription apparatus
(-> carboxylterminal domain)
CTD plays a role in transcrition regulation -> binds to mediatorPhosphorylation 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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Eukaryotic transcription products (from RNA polymerase II) are processed
Capping 5’ end
Polyadenylation of 3’ end
7-methylguanylate end
triphosphate
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RNA editing
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Splicing
Anemia: defect synthesis of
hemoglobin
Mutations affecting splice sites
cause around 15% of all genetic
diseasesCreates a new splice site
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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
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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
mRNA
Translation
Protein
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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
1st step: activation of AA by adenylation (Aminoacyl-AMP)
2nd step: linkage of AA to tRNA
Linkages either 2’ or 3’
1st step
2nd step
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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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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
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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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Ribosomes are Ribonucleoproteins
70S50S
30S
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Ribosomal RNAs (5S, 16S, 23S rRNA)
16S rRNA
secondary structure
tertiary 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 Elongation factor G
EF-Tu delivers aminoacyl-tRNA to Ribosomes
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)
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Protein Synthesis Termination by Release Factors
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Differences between Eukaryotic and Prokaryotic Protein Synthesis
Difference -> Translocation Initiation
Protein Interaction cirularize mRNA