How cells read the genome: from DNA to protein

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How cells read the genome from DNA to protein

• Chapter 6

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• Genes can be expressed with different efficiencies

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From DNA to mRNA
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• RNA Polymerase

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• Direction of RNA polymerase movement determines
  sequence of mRNA
• Direction determined by orientation of promoter
  sequence
• Template read in 3 to 5 direction

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• Genes are transcribed from both strands of the
  DNA
• Promoter sequence orientation determines direction

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• Initiation of transcription in eucaryote
• Multiple transcription factors (TFIIs) involved

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Consensus sequences in eukaryotic promoter regions
TATA binding protein (TBP)
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• Eukaryotes
• Introns must be spliced out
• 5-capping
• 3-polyadenylation
• Procaryotes
• Much simpler

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• A comparison of procaryotic and eucaryotic mRNA
  structures

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• 5Cap
• signals end of transcript
• identifies this as an mRNA
• is important for translation
• is first modification

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RNA factory
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• Eukaryotes
• Introns must be spliced out
• 5-capping
• 3-polyadenylation
• Procaryotes
• Much simpler

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Splicing of exons and introns
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Alternate splicing

• Alternate splicing of ?-tropomyosin gene
• Red arrows denote sites of cleavage and poly-A
  addition

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Splicing sites- AG-GU Branch point - A
R is A or G Y is C or U (polypyrimidine tract)
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• snRNPs
• snurps
• Spliceosome

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Splicing sites- AG-GU Branch point - A
R is A or G Y is C or U (polypyrimidine tract)
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Possible splicing errors
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?-thalassemia can result from splicing errors
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• Exon sizes quite uniform
• lt100-300bp
• Intron sizes more variable
• lt100-gt30,000bp

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• Eukaryotes
• Introns must be spliced out
• 5-capping
• 3-polyadenylation
• Procaryotes
• Much simpler

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• Signals mRNA for translocation and translation
• CstF-Cleavage stimulation factor F
• CPSF-cleavage and polyadenylation stimulation
  factor

• Poly-Adenylation

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Transport of mRNA through the nuclear pore complex
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• Export from the nucleus

hnRNP- heterogeneous nuclear ribonuclear proteins
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From mRNA to protein
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The Genetic Code mRNA
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Three possible reading frames on mRNA
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tRNA
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• tRNA modified with removal of intron (blue)
  before exit from nucleus
• tRNA endonuclease/ tRNA ligase

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W
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Amino acid activation of tRNA by aminoacyl tRNA
synthetase

• 2 step process
• Adenylated amino acid formation
• Linkage to tRNA

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Genetic code translated by two adaptor systems

• 1. Aminoacyl tRNA synthetase

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Genetic code translated by two adaptor systems

• 2. tRNA to mRNA hybridization
• Error in either step would result in wrong amino
  acid being incorporated into protein

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Hydrolytic editing ensures accuracy
tRNA synthetase
Correct amino acid cannot enter the editing site
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Incorporation of an amino acid into a protein
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Ribosomes
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• The Nucleolus
• Site of ribosome and other ribonucleoprotein
  synthesis
• Ribosomes must be exported from the nucleus

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• Meanwhile, back at the nucleus!

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Initiation of protein synthesis

• Eucaryotic initiation factors eIF-2, eIF-4G and
  eIF-4E
• Initiator tRNA (methionine)
• mRNA loop structure must be present before
  translation begins

EF-Tu-like GTP binding protein!
Fig 6-72
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Initiation of protein synthesis
Fig 6-72
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Initiation of protein synthesis in a
prokaryote Dont show
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Protein synthesis

• Involved
• Elongation Factor Tu
• Elongation Factor G

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Translation
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Detailed view of translation
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Termination

• Release factor(eRF1)

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Polyribosome-ensures full length mRNA in
eucaryotes
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Nonsense mediated decay (NMD)Eukaryotic quality
control system
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Rescue of ribosome stuck on a broken RNA-
Prokaryotic quality control system
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Creation of a functional protein
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Molten globule
Cytochrome b562
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Co-translation and folding
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Chaperones
Cytochrome b562
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Heat-shock proteins 70 (hsp70)
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Heat-shock proteins 60 (hsp60)
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Proteasome
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Breakdown of tagged protein
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Ubiquitin
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The marking of proteins with ubiquitin chains
Step 1 primes the E2/E3 ligase with
ubiquitin Step 2 adds ubiquitin to target
protein on lysine residue.
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• Ways to induce degradation of a specific protein
• Activation of a specific E3 molecule
  (e.g.anaphase
• promoting complex)

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Ways to induce degradation of a specific
protein b. Create an exposed degradation signal
on target
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Proteasome
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Prions

• Mad Cow disease (Bovine spongiform
  encephalopathy- BSE)
• Creutzfeldt-Jacob Disease (CJD) in humans
• Scrapie in Sheep