From chromosome to gene

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From chromosome to gene

• Eukaryotes contain a nucleus in which the
  chromosomes are located
• DNA
• 4 bases AT and C?G
• double helix with the bases as steps and a
  sugar-phosphate backbone
• Packaging is required to get all the DNA inside
  the nucleus
• double helix
• nucleosomes histone-DNA complex
• 10 nm fiber string of nucleosomes
• 30 nm fiber coil of string of nucleosomes
• DNA fiber attached to protein matrix at MARs
  formation of loops.
• A chromosome is a structure made of DNA and
  proteins. The metaphase chromosome represents
  most compact form of packaging
• Euchromatin open structure active genes
• Heterochromatin highly packaged structure
  inactive genes

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• A, B, Z conformation of DNA
• the B-conformation is the typical Watson-Crick
  double helix.
• the A-conformation occurs when DNA is dehydrated
• the Z-conformation is formed as a result of a
  certain base-order this is a left handed helix.
• DNA can bend and kink

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DNA
nucleosome
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30 nm fiber
10 nm fiber
5
Loops of histone depleted DNA
Protein matrix
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• Overview of gene expression in plants

exon
5
3
intron
promoter
AATAA
transcription
ATG
STOP
AAAAn
splicing
3UTR
5UTR
ORF
AAAAn
processing
nuclear export
mRNA degradation
translation, post-translational modification,
folding
active protein
protein degradation
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Transcription

• In eukaryotes transcription is carried out by
  three different RNA polymerases
• RNA polymerase I 28S, 5.8S and 18S rRNA
• RNA polymerase II protein coding genes and most
  snRNAs
• RNA polymerase III 5S rRNA, tRNA, U6 snRNA
• Transcription initiation. Binding of RNA
  polymerase II to the promoter region of the gene.
  This is mediated by the TBP (TATA-binding
  protein) and at least 12 TAFs (TATA-binding
  protein Associated Factors), together called
  TFIID. The TATA-box is a sequence found around
  pos. 25 in the promoter. A number of additional
  transcription factors (TFIIA, B, E, F, H) are
  involved in stabilizing the complex (A),
  recruiting RNA polymerase II (B, F), opening the
  DNA helix and phosphorylating the C-terminal
  domain of RNA Pol II (H). Phosphorylation allows
  the polymerase to leave the initiation complex.
• 2. Capping. After initiation of transcription
  by RNA pol II the pre-mRNA is capped at the 5
  end. The cap results from a GTP molecule
  reacting with the 5 end of the mRNA to give a
  5-5 bond. This is catalyzed by guanylyl
  transferase. The N7 position of the G residue is
  then methylated (cap 0) by guanine
  methyltransferase, but the 3 OH group of the
  first two bases of the mRNA may also be
  methylated (cap1 and cap2).
• 3. Elongation. Transcription of eukaryotic DNA
  can take a long time (up to 20 hrs) due to the
  length of the genes (introns!) and the presence
  of histones. Elongation factors stabilize the
  transcription complex.

focus
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• 4. Termination and poly-adenylation. A CPSF
  (Cleavage and Poly-adenylation Specificty Factor)
  is recruited at the initiation of transcription,
  identifies the poly-adenylation sequence (AAUAA),
  changes conformation, which then results in the
  termination of transcription. After termination
  of transcription a poly-A tail (approx. 250
  adenosine residues) is added by poly(A)
  polymerase. Poly-adenylation is mediated by the
  binding of a CPSF, and a CstF (Cleavage
  stimulation Factor). A poly-adenylate binding
  protein helps the polymerase add A-residues and
  stabilizes the poly-A tail after synthesis. The
  exact function of the poly-A tail is unknown.
• Intron splicing. Exons are coding DNA sequences,
  while introns are non-coding. After
  transcription, the exons need to be joined to
  generate the amino-acid coding mRNA. The purpose
  of introns is not entirely clear. The most common
  type of intron is the GU-AG intron, which refers
  to the 5 en 3 ends of the intron sequence. A
  poly-pyrymidine tract is found just upstream of
  the 3 end.
• Intron splicing is mediated by small nuclear
  RNAs (U1, U2, U4, U5 and U6 snRNAs) that
  associate with proteins to form snRNPs (small
  nuclear ribonucleoproteins). Splicing occurs in
  three steps
• Formation of a commitment complex involving
  U1-snRNP (binds to 5 splice site) and several
  protein factors
• Formation of the pre-spliceosome complex, after
  binding of U2-snRNP
• Formation of the spliceosome, after binding of U5
  and U4/6 snRNPs.
• Splicing is a multi-step process that involves a
  cut at the 5 end of the intron, formation of a
  lariat structure, a cut at the 3 end of the
  intron, joining of the exons and debranching of
  the lariat. The snRNPs mediate these different
  steps.
• 6. Nuclear export. After the splicing is
  complete, the mRNA is transported to the
  cytoplasm where it will be translated.

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TATA-binding protein
Stabilizing complex
Recruitment of polymerase
Recruitment of polymerase
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(No Transcript)
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Termination and poly-adenylation
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Splicing- mediated by snRNPs