the central dogma of molecular biology by e. börje lindström this learning object has been funded...
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The Central Dogma of Molecular Biology
by
E. Börje Lindström
This learning object has been funded by the European Commissions FP6 BioMinE project
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The flow of information
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DNA molecule• General structure: - double stranded
- complementary
- helical
- antiparallel
• Strands: - backbone of alternating phosphate and
deoxyribos units
- four different bases; adenine (A), guanine (G), cytosine ( C ), and thymine (T).
• Double helix: - due to base pairing: A=T and GC
• Major and Minor groove
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DNA molecule, cont.
• Size: - units: kilobase (kb) or kilobase pairs (kb pairs)
- E. coli chromosome 4 700 kb pairs
• Form: - closed chromosome molecule (in bacteria)
- 1 mm long packing problem in bacteria
- solved by supercoiling
• DNA binding proteins:
Un-specific: - histones
Specific: -Repressors
- RNA polymerase
- restriction enzymes
- modification enzymes
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DNA molecule, cont.
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DNA molecule, cont.
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DNA replication
• Semi conservative: -new DNA molecules contain:
1 old strand and
1 new strand
• use a ’template’: - one of the strand is used
• ’primers’: -usually a piece of RNA
- DNA-polymerase unable to start replication
General
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Initiation of replication• Start point: -only one (1) on the chromosome (300 bp)
- origin (ori)
ori
• bidirectional: - both directions
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Synthesis of DNA (replication)• several enzymes involved (~ 20 pc)
- DNA helicase Unwinding the molecule
- DNA gyrase (topoisemerase II)
Open up (cut) the strands
- DNA-binding enzymes Protect ss-DNA from nucleases
- Primase Synthesises the RNA primer
- DNA-plymerase III Synthesis in direction 5’3’
There are 3 enz. in E. coli; pol I, II and III
- DNA-plymerase I Removes the primer
Repair any missing bp in DNA
- DNA ligase Makes a phospho-di-ester bond (glueing)
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Synthesis of DNA, cont.• ’leading’ and ’lagging’ strands:
- leading: continous synthesis
- lagging: dis-continous synthesis
• proof-reading: - checking if any mitakes has been made
- pol. III removes the wrong nucleotides (3’ 5’)
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Figures, DNA replication
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RNA transcriptionThree types of RNA: • mRNA (genetical)
• tRNA (aa-carrier)
• rRNA (structural)
Structure: -ss-stranded (internal ds secundary structures)
- ribose
- four different bases; adenine (A), guanine (G), cytosine ( C ), and uracile (U).
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Synthesis of RNA• ds DNA is the template: - only one of the strands
• RNA polymerase: - consists of four different subunits
- 2’
- 2’ = core enzyme
- recognises the start site
• Direction of synthesis: - 3’5’
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Start and stop of RNA synthesis• Where is the start ?
- Note! No primers necessary!
- The polymerase binds to the promoter
- recognises and attaches to the promoter region
- ds-DNA opens up and the synthesis starts
- is detached and the core enzyme continues
• Where does the synthesis stop?
-termination at special DNA-sequenses, terminators
- inverted repeates in DNA ’stem-loop’-structures in RNA
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PromotersA sequence in DNA upstreams a structural gene:
• -10 sequence Pribnow box
• Strong promoters bind effective
SG
-35bp -10bp
P
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mRNA
• Short half-time
• Polycistronic (in bacteria) - information from several structural genes
• Definitions:
- operator (O): a gene that can be effected by a repressor protein
- operon: structural genes with the same repressor
SG1OP SG2 SG3
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TranslationNecessary substances:
• mRNA
• ribosomes
• tRNA + aa tRNAaa (attached aa)
• different factors
• enzymes
• energy
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tRNA
• DNA-genes: - Linear tRNA form (primary)
- cloverleaf structure (secundary)
• Two peoperties: - binds aa (enzymatic)
- binds to mRNA (codon) with its anti-codon
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tRNA, cont.
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Synthesis of proteinsA four (4) step process: • Initiation
• Elongation
• Termination-release
• Peptide folding
• Initiation: -a complex of
- 30S subunit,
- f-meth-tRNA, (start codon AUG in mRNA)
- mRNA and
- initiation factors are formed
• Shine-Delgarno sequence -3-9 bases in mRNA
- complementary to 16S rRNA
- addition of 50S subunit
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Synthesis of proteins, cont.• Elongation: -several elongation factors are needed
- Next aa-tRNA is added to the A-site (ribosome)
- a peptide bond is created
- the peptide is moved to the A-site
- translocation to the P-site during
- movement of the ribosome forward
- a free A-site is created …
-Etc.
• polysomes: - mRNA with several ribosomes
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Synthesis of proteins, cont.• Termination: -stop codes in mRNA
- UAA, UAG and UGA; nonsence codes
- no tRNA for these codes exist
- release factors RF1-3 release the protein
- the ribosomes disintegrate
• The genetic code: - in mRNA
• 3 bases - 1 aa
• 43 = combinations -but only 24 aa
- degenerated code
- the aa has several codes
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Reading frame• Open reading frame (ORF): - a gene
AUG UAGS D-G
• Codon usage: -The code (tripletts) does not mean the same in all organisms
- The mRNA or ORF give different products
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The wobble concept