02 26 15 Chbe 310
Jan 25, 2016
DNA RNA PROTEINS
transcription
Replication
translation
Universality of the central dogma: implications in biotech?
Control:
6
8
• Double helix structure of DNA
• complementary strands
• DNA replication is semiconservative
Meselson and Stahl experiment
Bacteria are grown in medium containing only the “heavy isotope” of nitrogen (15N)
Bacteria are then transferred to 14N medium for several generations In each generation, buoyant density centrifugation (in CsCl) was performed on a DNA extract from the bacteria. Heavier DNA molecules spin down further in the gradient tube.
DNA Replica9on
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• Polymerase: DNA polymerase vs RNA polymerase
• DNA-RNA does not stay hybrid (multiple mRNA at the same time)
• Proofreading mechanisms:
Transcription is less accurate, lack of extensive proofreading mechanisms: why?
DNA replica9on vs RNA transcrip9on
DNA vs RNA
• RNA, DNA: linear polymers, phosphodiester bond
DNA: deoxyribose RNA: ribose DNA: AGCT RNA: AGCU Errors: DNA Pol 1/107 errors: RNA Pol 1/104
DNA more stable RNA less stable – shorter half life
Consensus sequence = affinity of recognition between DNA and polymerase = strength of promoter = yield of transcription
PROMOTER
Site for termina9on of transcrip9on
Site for ini9a9on of transcrip9on by RNA polymerase The promoter regulates
where, when and to what level a gene is expressed
TATAAT TTGACA
-35 (17-19bp) -10 +1
START STOP
ATG$It$is$the$only$start$codon$and$corresponds$to$Methionine$
TAA$TAG$TGA$Signal$stop$of$transla8on$
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mRNA messenger
tRNA transfer (specifies AA)
rRNA ribosomal (peptide bond)
PROTEINS Structural
Catalytic
Transport
Regulatory
Protective
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ORF = OPEN READING FRAME
START STOP
AUG
Only codon for Met preceded by purine reach sequence at START
UAA
UAG
UGA
Typically bound by release factors
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Post-‐transla9onal events
FOLDING
• Chaperones / degradation
• Formation of IB in bioprocesses
SIGNAL SEQUENCE = 20-25 aa are clipped off
• cytoplasm / periplasm
• ER / secretion