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
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Prokaryotic Cells
http://atlas.arabslab.com
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Comparison to Eukaryotic Cells
http://www.cod.edu/people/faculty/fancher/ProkEuk.htm
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Biochemistry
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Nucleic Acids (DNA, RNA)
Proteins
Lipids
Carbohydrates
Small Molecules
Ions
Replication
DNA
Transcription
RNA
Translation
Protein
Metabolism
Control
Structural
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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
Control
David S. Goodsell (Scripps)
Structural
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DNA
Replication
DNA
Transcription
RNA
Translation
Protein
Metabolism
Control
Structural
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DNA
Replication
DNA
Transcription
RNA
Translation
Protein
Metabolism
Control
Structural
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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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Smallest Genome – was in 1999
}
Single Gene
One of the smallest Genomes: Mycoplasma genitalium (Small parasitic bacterium)
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Smallest Genome
Total genes:
Protein coding genes:
tRNA and rRNA:
521
482
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
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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:
Pyruvate:
ATP:
Ca ions:
Fe ions:
350,000
370,000
2,000,000
2,300,000
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)
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E. Coli Statistics
E coli has approximately 4300 protein coding genes.
Protein abundance per cell:
ATP Dependent helicase: 104
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
Source: Protein abundance profiling of the Escherichia coli cytosol.
BMC Genomics 2008, 9:102. Ishihama et al.
Krebs Cycle
Malate Dehydrogenase: 3,390
Citrate Synthase:
1,360
Aconitase:
1630
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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
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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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RNA Polymerase Stops at a
Terminator
Changes in the terminator sequence
can change the efficiency of RNA
polymerase stopping. If the gene is
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
100%
Promoter
Gene B
60%
Terminator
Gene C
30%
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Operators – Regulating Expression
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Gene Regulation
lac Operon
Metabolic Enzyme (output)
Promoter
Promoter
Operator
lacZ codes for β-galactosidase.
lacY codes for β-galactoside permease.
Sugar in Medium
Relative βgalactosidase
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.
Lactose
Allolactose
Allolactose is the natural inducer
β-galactosidase, as well as hydrolyze Lactose to
Glucose and galactose, it can also catalyze the
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
LI
Lac Genes
E
3
LE
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Lac Operon Wiring Diagram
Operator
Lac Genes
4
LI
E
LE
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Lac Operon Wiring Diagram
Operator
Lac Genes
4
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
4
LI
2
1
E
3
LE
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Lac Operon Wiring Diagram
Operator
LI
LE
Lac Genes
E
p = defn cell
E -> $W; k1*E;
//
LI -> $W; k2*E*LI;
//
LE -> LI; k3*E*LE;
//
$G -> E; Eo + k4*LI^n/(Km1
end;
p.n
p.LE
p.Eo
p.Km1
p.k1
p.k3
p.E
=
=
=
=
=
=
=
Enzyme degradation
Lactose metabolism
Lactose transport
+ LI^n); // Lac expression
2;
5;
0.01;
0.5;
0.1; p.k2 = 0.1;
0.1; p.k4 = 0.1;
0;
p.LI = 0;
m = p.sim.eval (0, 120, 500, [<p.Time>, <p.E>, <p.LI>, <p.LE>]);
graph (m);
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Lac Operon Wiring Diagram
Operator
LI
Lac Genes
E
LE
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Lac Operon Wiring Diagram
Operator
LI
Lac Genes
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
Operators
Start Codon
Stop Codon
Gene
Promoter
RBS
Terminator
5’- UTR
All these regions are potentially engineerable.
RBS = Ribosome Binding Site
UTR = Untranslated Region
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Genetic Networks can get Complicated
http://www.biotapestry.org/
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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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