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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