02  26  15  

Chbe  310    

X1                                                        X2                                                        X3                                                      X4                                              X5      E1                                                            E2                                                        E3                                                  E4    

Metabolic  Regula9on  

2  

Metabolic  Regula9on  

3  

Problem  

How  Cells  Work  

Shuler,  Ch  4  

5  

DNA RNA PROTEINS

transcription

Replication

translation

Universality of the central dogma: implications in biotech?

Control:

6  

7  

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  

9  

DNA  Replica9on  

DNA  polymeriza9on  occurs  in  the  5’à  3’  direc9on  

DNA  Replica9on  

10  

DNA  Replica9on  

12  

13  

Codon  usage  

14  

DNA  Replica9on  

15  

DNA  Replica9on:  elonga9on  

16  

Informa9on  transfer:  from  DNA  to  proteins  

17  

18

Proofreading  ac9vity  

19

•  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$

Protein-­‐DNA  Interac3ons  are  key  to  gene  expression  

RNA  transcrip9on:  mRNA  synthesis  

23  

Eukaryo9c  cells  and  mRNA  synthesis  

splicing  

24  

Transla9on  

http://www.youtube.com/watch?v=0248WyghCjc&feature=related 25  25

26

mRNA messenger

tRNA transfer (specifies AA)

rRNA ribosomal (peptide bond)

PROTEINS Structural

Catalytic

Transport

Regulatory

Protective

24

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

27

Transla9on  

5’                                                                                          3’   N                                                                                          C  

Transcrip9on  of  two  genes  

(Which  is  the  DNA  and  which  the  mRNA?)  

29  

Post-­‐transla9onal  events  

FOLDING

•  Chaperones / degradation

•  Formation of IB in bioprocesses

SIGNAL SEQUENCE = 20-25 aa are clipped off

•  cytoplasm / periplasm

•  ER / secretion

Post-­‐transla9onal  modifica9ons  

GLYCOSILATION

•  Differences btw eukaryotes and prokaryotes

•  AA consensus sequence

DISULFIDE BONDS FORMATION

•  Differences btw eukaryotes (ER) and prokaryotes (periplasm)