02

26 15
Chbe 310

Metabolic Regula9on

E
E
E3 E4
X1 1 X 2 2 X
3 X4 X5

Metabolic Regula9on

Problem

How Cells Work

Shuler, Ch 4

DNA

RNA
transcription

PROTEINS
translation

Replication

Universality of the central dogma: implications in biotech?

Control:

DNA Replica9on
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.
8

DNA Replica9on
DNA polymeriza9on occurs in the 5 3 direc9on

DNA Replica9on

10

DNA Replica9on

12

Codon usage

13

DNA Replica9on

14

15

DNA Replica9on: elonga9on

16

Informa9on transfer: from DNA to proteins

17

Proofreading ac9vity

18

DNA replica9on vs RNA transcrip9on

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?

19

DNA vs RNA
RNA, DNA: linear polymers, phosphodiester bond
DNA: deoxyribose
DNA: AGCT
Errors: DNA Pol 1/107
DNA more stable

RNA: ribose
RNA: AGCU
errors: RNA Pol 1/104
RNA less stable shorter
half life

PROMOTER

The promoter regulates

where, when and to what
level a gene is expressed

Site for ini9a9on of

transcrip9on by RNA
polymerase

Site for termina9on

of transcrip9on

Consensus sequence = affinity of recognition between DNA and polymerase

= strength of promoter = yield of transcription
START
-35
TATAAT

(17-19bp) -10
TTGACA

+1
ATG$
It$is$the$only$
start$codon$and$
corresponds$to$
Methionine$

STOP

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

mRNA messenger
PROTEINS

Structural
Catalytic
Transport
Regulatory
Protective

tRNA

transfer (specifies AA)

rRNA

ribosomal (peptide bond)

ORF = OPEN READING FRAME

T
AR
T
S

OP
ST

AUG

UAA

Only codon for Met

preceded by purine
reach sequence at
START

UAG
UGA
Typically bound by
release factors

26

24

Transla9on

5 3

N C

27

Transcrip9on of two genes

(Which is the DNA and which the mRNA?)

Post-transla9onal events

FOLDING
Chaperones / degradation
Formation of IB in bioprocesses
SIGNAL SEQUENCE = 20-25 aa are clipped off
cytoplasm / periplasm
ER / secretion
29

Post-transla9onal modica9ons

GLYCOSILATION
Differences btw eukaryotes and prokaryotes
AA consensus sequence

DISULFIDE BONDS FORMATION

Differences btw eukaryotes (ER) and prokaryotes
(periplasm)