Prokaryotic Biology and Genetics

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Prokaryotic Cells

1 .Bacteria lack membrane bound nuclei

2. DNA is circular
3. No complex internal organelles

3 um

http://www.ucmp.berkeley.edu/bacteria/bacteriamm.html 2
Prokaryotic Cells

http://atlas.arabslab.com
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Comparison to Eukaryotic Cells

http://www.cod.edu/people/faculty/fancher/ProkEuk.htm 4
 Biochemistry
• Nucleic Acids (DNA, RNA)
• Proteins Replication
DNA
• Lipids
Transcription
• Carbohydrates RNA
• Small Molecules Translation
• Ions Protein

Metabolism Structural
Control

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Biochemistry
Average spacing between proteins:
7 nm/molecule

Diameter of a protein: 5 nm

Replication
DNA
Transcription

RNA
Translation

Protein

Metabolism Structural
Control
David S. Goodsell (Scripps) 6
DNA
Replication
DNA
Transcription

RNA
Translation

Protein

Metabolism Structural
Control

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DNA
Replication
DNA
Transcription

RNA
Translation

Protein

Metabolism Structural
Control

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DNA

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RNA

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RNA

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 RNA Polymerase

mRNA
5’ end

http://mgl.scripps.edu/people/goodsell/pdb/pdb40/pdb40_1.html

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 Single Gene
Smallest Genome – was in 1999
}

One of the smallest Genomes: Mycoplasma genitalium (Small parasitic bacterium) 13

 Smallest Genome
Total genes: 521
Protein coding genes: 482
tRNA and rRNA: 39

This genome is of interest to synthetic biology because Craig Venter wants to use
this organism as the basis for a minimal organism for genetic engineering.

Venter’s group has removed roughly 101 genes and the organism is still viable,
the idea then is to patent the minimal set of genes required for life.

PNAS (2006) 103, 425--430

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 Gene Function

The complexity of simplicity

Scott N Peterson and Claire M Fraser
Genome Biol. 2001;2(2):COMMENT2002. Epub 2001 Feb 8.

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 But the real prize goes to….
The 160-Kilobase Genome of the
Bacterial Endosymbiont Carsonella

Atsushi Nakabachi, Atsushi

Yamashita, Hidehiro Toh, Hajime
Ishikawa, Helen E. Dunbar, Nancy
A. Moran, and Masahira Hattori

(13 October 2006)

Science 314 (5797), 267.

160-Kilobase Genome of the Bacterial Endosymbiont Carsonella

Symbiont of sap sucking PSYLLIDS or ‘jumping plant lice’ ~182 genes 16
E. Coli Statistics
Length: 2 to 3 um
Diameter: 1 um
Generation time: 20 to 30 mins

Translation rate: 40 aa/sec

Transcription rate: 70 nt/sec

Number of ribosomes per cell : 18,000

Small Molecules/Ions per cell:

Alanine: 350,000
Pyruvate: 370,000
ATP: 2,000,000
Ca ions: 2,300,000
Fe ions: 7,000,000

Data from: http://bionumbers.hms.harvard.edu

http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi

David S. Goodsell (Scripps) 17

 E. Coli Statistics Protein abundance per cell:

ATP Dependent helicase: 104

E coli has approximately 4300 protein coding genes.
LacI repressor: 10 to 50 molecules
LacZ (galactosidase) : 5000

CheA kinase (chemotaxis): 4,500

CheB (Feedback): 240
CheY (Motor signal): 8,200
Chemoreceptors: 15,000

Glycolysis
Phosphofructokinase: 1,550
Pyruvate Kinase: 11,000
Enolase: 55,800
Phosphoglycerate kinase: 124,000

Krebs Cycle
Malate Dehydrogenase: 3,390
Citrate Synthase: 1,360
Source: Protein abundance profiling of the Escherichia coli cytosol.
BMC Genomics 2008, 9:102. Ishihama et al.
Aconitase: 1630 18
Circular Chromosome in E. coli

Page 231

Most Prokaryotic DNA is circular. Gene are

located on both strands of the DNA. Genes
on the outside are transcribed clockwise
and those on the inside anticlockwise.

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EcoCyc: http://ecocyc.org/

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 E. coli Gene Structure

Stop codon
(TAG, TAA, TGA)

Start codon

Page 134 21
 RNA Polymerase Binds to Promoters
mRNA
Changes in the promoter sequence
can change the efficiency of RNA
polymerase binding to the DNA.

The promoter is therefore a site

which can be engineered.

http://mgl.scripps.edu/people/goodsell/pdb/pdb40/pdb40_1.html

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 Strong and Weak Promoters
The strength of a promote is one of the factors which
determines the rate of transcription.

Strong Promoter. The recA promoter is a strong promoter.

TTGATA -- 16 -- TATAAT
TTGACA -- 17 -- TATAAT Most common Promoter
(Consensus sequence)
It differs from the averaged promoter sequence by one nucleotide and on base
pair in the spacer region.

Weak Promoter. The araBAD promoter is a weak promoter.

CTGACG -- 18 -- TACTGT
TTGACA -- 17 -- TATAAT

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 Changes in the terminator sequence
RNA Polymerase Stops at a can change the efficiency of RNA
polymerase stopping. If the gene is
Terminator part of an operon, terminators can
modulate relative expression levels of
the different genes in the operon.

The terminator is therefore a site

which can be engineered.

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 Operon Structure

Gene A Gene B Gene C

100% 60% 30%

Promoter Terminator

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Operators – Regulating Expression

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Gene Regulation
lac Operon

Metabolic Enzyme (output)

Promoter
Promoter
Operator
Sugar in Medium Relative β-
galactosidase
lacZ codes for β-galactosidase.
lacY codes for β-galactoside permease. Glucose 1
Glucose + lactose 50
Lactose 2500
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 Gene Regulation
lac Operon

Lac repressor

Metabolic Enzyme (output)

Promoter Promoter

Operator
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Gene Regulation
lac Operon
Lactose is galactose-(β1->4)-glucose, whereas
allolactose is galactose-(β1->6)-glucose.

β-galactosidase, as well as hydrolyze Lactose to

Lactose Allolactose
Glucose and galactose, it can also catalyze the
Allolactose is the natural inducer Conversion of Lactose to Allolactose.
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 LacI Repressor

lacI is a tetramer (x4)

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LacI binding to Promoter

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 Lac Operon Wiring Diagram

lacI Operator β-galactosidase β-galactoside permease

Allolactose

Lactose Internal

Lactose External
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 Lac Operon Wiring Diagram
Operator Lac Genes

Lactose
Internal

Lactose
External

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 Lac Operon Wiring Diagram
Operator Lac Genes

1
LI E

LE

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 Lac Operon Wiring Diagram
Operator Lac Genes

2
LI E

LE

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 Lac Operon Wiring Diagram
Operator Lac Genes

LI E
3

LE

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 Lac Operon Wiring Diagram
Operator Lac Genes

LI E

LE

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 Lac Operon Wiring Diagram
Operator Lac Genes

LI E
Two state variables: LI and E
Seven Parameters: k1, k2, k3, k4, Eo, n, Km
LE
What about LE ?

Depends on the simulation. If external lactose

is assumed to be fixed then LE is a boundary species
else it is assumed to be another state variable.
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 Lac Operon Wiring Diagram
Operator Lac Genes

2 1
LI E
3

LE

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 Lac Operon Wiring Diagram
Operator Lac Genes

LI E p = defn cell
E -> $W; k1*E; // Enzyme degradation
LI -> $W; k2*E*LI; // Lactose metabolism
LE -> LI; k3*E*LE; // Lactose transport
$G -> E; Eo + k4*LI^n/(Km1 + LI^n); // Lac expression
end;

LE p.n = 2;
p.LE = 5;
p.Eo = 0.01;
p.Km1 = 0.5;
p.k1 = 0.1; p.k2 = 0.1;
p.k3 = 0.1; p.k4 = 0.1;
p.E = 0; p.LI = 0;

m = p.sim.eval (0, 120, 500, [<p.Time>, <p.E>, <p.LI>, <p.LE>]);

graph (m); 40
 Lac Operon Wiring Diagram
Operator Lac Genes

LI E

LE

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 Lac Operon Wiring Diagram
Operator Lac Genes

LI E

LE

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 Where did the gene expression rate
law come from?

Fit this data to the above equation, this yields a workable equation

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Ribosome Binding Sites

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 In summary

Start Codon Stop Codon

Operators
Gene

Promoter RBS Terminator

5’- UTR

All these regions are potentially engineerable.

RBS = Ribosome Binding Site

UTR = Untranslated Region 45
 Genetic Networks can get Complicated

http://www.biotapestry.org/ 46
Protein Signaling Maps

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 Protein Signaling Maps
20% of the human protein-coding
genes encode components of signaling
pathways, including transmembrane
proteins, guanine-nucleotide binding
proteins (G proteins), kinases,
phosphatases and proteases.

The identification of 518 putative

protein kinase genes and 130 protein
phosphatases suggests that
reversible protein phosphorylation is
a central regulatory element of most
cellular functions.

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Metabolic Maps

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