Transcription and splicing machinery
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DNA  primary mRNA  mature mRNA. Transcription + Processing. Transcription and Splicing machinery. Prokaryotic and Eukaryotic RNA Polymerases are similar in shape. Sigma ( σ ) subunit missing. -> Different number of subunits. Recognizes the promoter site (-10 box + -35 box).

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Transcription and Splicing machinery

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Transcription and splicing machinery

DNA  primary mRNA  mature mRNA

Transcription + Processing

Transcription and Splicing machinery


Transcription and splicing machinery

Prokaryotic and Eukaryotic RNA Polymerases are similar in shape

Sigma (σ) subunit missing

-> Different number of subunits


Transcription and splicing machinery

Recognizes the promoter site (-10 box + -35 box)


Transcription and splicing machinery

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+


Transcription and splicing machinery

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


Transcription and splicing machinery

Prokaryotic promoter sites

-35 -10 +1

5’-----TTGACA--------------TATAAT---------start site----3’

σ subunit


Transcription and splicing machinery

Prokaryotic promoter sites

σ subunit interacts with

-10 box and -35 box


Transcription and splicing machinery

Alternative E. coli promoters

Stanard Promoter -> σ70

Heat shock promoter -> σ32

N-starvation promoter -> σ54


Transcription and splicing machinery

Footprinting


Transcription and splicing machinery

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


Transcription and splicing machinery

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


Transcription and splicing machinery

Transcription bubble

First Nucleotide is pppG or pppA -> Transcription start


Transcription and splicing machinery

RNA-DNA hybrid separation

RNA polymerase forces the separation of the RNA-DNA hybrid


Transcription and splicing machinery

Transcription Termination

Rho independent termination

Termination by Rho protein

-> RNA polymerase pauses after production of hairpin

-> RNA-DNA hybrid of hairpin is unstable

=> RNA falls off

Rho interacts with RNA polymerase -> breaks the RNA-DNA hybrid helix -> functions as a helicase


Transcription and splicing machinery

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)


Transcription and splicing machinery

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


Transcription and splicing machinery

tRNA transcript processing

Modification: 1. Cleavage of primary transcript by Ribonuclease III

2. Addition of nucleotides at 3’ end (CCA)

3. Unusual bases


Transcription and splicing machinery

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


Transcription and splicing machinery

Transcription and Translation in Prokaryotes and Eukaryotes


Transcription and splicing machinery

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)


Transcription and splicing machinery

Different Eukaryotic RNA Polymerase promoters

Inr -> Initiator element

(found at transcription start)

DPE -> downstream core promoter element


Transcription and splicing machinery

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


Transcription and splicing machinery

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.


Transcription and splicing machinery

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


Transcription and splicing machinery

Eukaryotic Transcription Initiation Complex


Transcription and splicing machinery

Regulation of Transcription


Transcription and splicing machinery

Packaging of Eukaryotic chromosomal DNA


Transcription and splicing machinery

Transcription Initiation


Transcription and splicing machinery

Gene “Off”

Gene “On”


Transcription and splicing machinery

Eukaryotic transcription products (from RNA polymerase II) are processed

triphosphate

7-methylguanylate

end

Polyadenylation of 3’ end

Capping 5’ end


Transcription and splicing machinery

RNA editing


Transcription and splicing machinery

Splicing

Anemia: defect synthesis of hemoglobin

Mutations affecting splice sites cause around 15% of all genetic diseases

Creates a new splice site


Transcription and splicing machinery

Small nuclear RNAs in spliceosomes catalyse the splicing


Transcription and splicing machinery

Spliceosome assembly

The catalytic center of the spliceosome


Transcription and splicing machinery

Alternative splicing


Transcription and splicing machinery

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.


Transcription and splicing machinery

Self-splicing mechanism


Transcription and splicing machinery

Ribosomal Factory

Protein

mRNA

Translation


Transcription and splicing machinery

Translation:

mRNA -> Protein


Transcription and splicing machinery

Peptide bond formation in Ribosomes


Transcription and splicing machinery

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


Transcription and splicing machinery

Aminoacyl-tRNA synthetases couple Amino acids to tRNA

Synthetases are highly specific for the amino acid (error rate 1 in 105)


Transcription and splicing machinery

Proofreading of Aminoacyl-tRNA Synthetases


Transcription and splicing machinery

Synthetases recognize the anticodon loops and acceptor stems of tRNA

Threonyl-tRNA synthetase complex

Class II synthetase

Glutaminyl-tRNA synthetase complex

Class I synthetase


Transcription and splicing machinery

Classification of Aminoacyl-tRNA synthetases

  • Synthetases recognize different faces of the tRNA molecule:

  • Class I acylates the 2’ OH group of the terminal adenosine of tRNA

  • Class II acylates the 3’ OH group of the terminal adenosine of tRNA

  • They bind ATP in different conformations

  • Most class I are monomeric, most class II are dimers


Transcription and splicing machinery

Ribosomes are Ribonucleoproteins

70S

50S

30S


Transcription and splicing machinery

Ribosomal RNAs (5S, 16S, 23S rRNA)

16S rRNA

tertiary structure

secondary structure


Transcription and splicing machinery

Ribosomal Protein L19 of the 50S subunit

Fits through some of the cavities within the 23S RNA


Transcription and splicing machinery

Protein synthesis in E. coli

Polysomes: Transcription and Translation happens at the same time

Direction of Transcription: 5’->3’

Direction of Translation: 5’->3’


Transcription and splicing machinery

Translational Initiation sites – Ribosome binding sites


Transcription and splicing machinery

Bacterial Protein synthesis is initiated by Formylmethionyl tRNA -> fMet


Transcription and splicing machinery

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


Transcription and splicing machinery

Polypeptide chain escape path

Polypeptide synthesis tunnel


Transcription and splicing machinery

Peptide bond formation


Transcription and splicing machinery

Some tRNAs recognize more than one codon -> wobble base


Transcription and splicing machinery

Elongation factor Tu

Elongation factor G

EF-G mediates translocation within the Ribosome

EF-Tu delivers aminoacyl-tRNA to Ribosomes


Transcription and splicing machinery

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


Transcription and splicing machinery

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)


Transcription and splicing machinery

Protein Synthesis Termination by Release Factors


Transcription and splicing machinery

Differences between Eukaryotic and Prokaryotic Protein Synthesis

Difference -> Translocation Initiation


Transcription and splicing machinery

Protein Interaction cirularize mRNA


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