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I. The Blueprint of Life: Structure of the Bacterial
Genome
• 4.3 Genetic Elements: Chromosomes and
Plasmids
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4.3 Genetic Elements: Chromosomes and
Plasmids
• Genome: entire complement of genes in cell or
virus
• Chromosome: main genetic element in
prokaryotes, usually a single piece of DNA
• Other genetic elements include virus genomes,
plasmids, transposable elements (jumping genes)
and in eukaryotes-genomes of organelles
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4.3 Genetic Elements: Chromosomes and
Plasmids
• Viruses may be either RNA or DNA
• Plasmids: replicate separately from chromosome
• Great majority are double-stranded
• Most are circular
• Generally beneficial for the cell (e.g., antibiotic
resistance)
• NOT extracellular, unlike viruses
• “autonomous”
• “episome”
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4.3 Genetic Elements: Chromosomes and
Plasmids
• Chromosome is a genetic element with
"housekeeping" genes
• Presence of essential genes is necessary for a genetic
element to be called a chromosome
• Plasmid is a genetic element that is expendable
and rarely contains genes for growth under all
conditions-extra genes
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4.3 Genetic Elements: Chromosomes and
Plasmids
• Transposable elements
• Segment of DNA that can move from one site to another
site on the same or a different DNA molecule
• Inserted into other DNA molecules
• Three main types:
• Insertion sequences (IS)
• Transposons (Tn)
• Certain viruses
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4.3 Genetic Elements: Chromosomes and
Plasmids
• The Escherichia coli chromosome
• Escherichia coli is a useful model organism for the
study of biochemistry, genetics, and bacterial
physiology
• The E. coli chromosome from strain MG1655 has
been mapped using a combination of biological
and biochemical methods (Figure 4.8)
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© 2015 Pearson Education, Inc.
Figure 4.8
4.3 Genetic Elements: Chromosomes and
Plasmids
• Some features of the E. coli chromosome
• Many genes encoding enzymes of a single biochemical
pathway are clustered into groups called operons
• Operons are equally distributed on both strands
• ~5 Mbp in size (entire chromosome)
• ~40% of predicted proteins are of unknown function
• Average protein contains ~300 amino acids
• Genes are close together
• Few repeated genes
• Few intervening sequences or introns
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4.3 Genetic Elements: Chromosomes and
Plasmids
• Plasmids: genetic elements that replicate
independently of the host chromosome (Figure 4.9)
• Small circular or linear DNA molecules
• Range in size from 1 kbp to >1 Mbp; typically less than
5% of the size of the chromosome
• Carry a variety of nonessential, but often very helpful,
genes
• Abundance (copy number) is variable: relaxed vs
stringent
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© 2015 Pearson Education, Inc.
Figure 4.9
4.3 Genetic Elements: Chromosomes and
Plasmids
• A cell can contain more than one plasmid
• Genetic information encoded on plasmids is not
essential for cell function under all conditions BUT
may confer a selective growth advantage under
certain conditions
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4.3 Genetic Elements: Chromosomes and
Plasmids
• R plasmids
• Resistance plasmids; confer resistance to antibiotics
and other growth inhibitors
(Figure 4.10)
• Widespread and well-studied group of plasmids
• Many code for conjugation functions: they can move into
a new cell through conjugation
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© 2015 Pearson Education, Inc.
Figure 4.10
4.3 Genetic Elements: Chromosomes and
Plasmids
• In several pathogenic bacteria, virulence
characteristics are encoded by plasmid genes
• Virulence factors
• Enable pathogen to colonize
• Enable pathogen to cause host damage
• Hemolysin
• Enterotoxin
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4.3 Genetic Elements: Chromosomes and
Plasmids
• Bacteriocins
• Proteins produced by bacteria that inhibit or kill closely
related species or even different strains of the same
species
• Colicin, nisin
• Genes encoding bacteriocins are often carried on
plasmids
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4.7 Transcription
• Transcription (DNA to RNA) is carried out by
RNA polymerase (Rpol) using DNA as template
with RNA chain growth in 5’ to 3’ direction.
• Transciption begins at a DNA sequence called a
promoter. Also requires sigma factor or start
factor.
• Transcription stops at specific sites called
transcription terminators
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RNA
polymerase
(core
enzyme)
Sigma recognizes
promoter and
initiation site.
Sigma
factor
5′
3′
3′
5′
Gene(s) to be transcribed
(light green strand)
Promoter region
Transcription begins;
sigma released. RNA
chain grows.
Sigma
5′
3′
3′
5′
5′
RNA
5′
3′
3′
5′
5′
Termination site
reached; chain
growth stops.
5′
3′
3′
5′
5′
Polymerase and RNA
released.
5′
3′
3′
5′
3′
5′
DNA
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Short transcripts
Longer transcripts
Figure 4.20
RNA polymerase
(core enzyme)
Transcription
5′
3′
3′
5′
Sigma
1.
2.
3.
4.
5.
6.
mRNA start
5′
3′
C T G T T G A C A A T T A A T T A T C C A AA T AG T T A A C T A G T A G G G A A G
C T A T T C C T G T GGA T A A C C A T G TG TAT TAG A G T T AG A A A A C A
T G G T T C C AA AA T CGCC T T T T G CT GTA TA T A C T C AC A G C A T A
T T T T T G A GT TGTG T A T A A CC C CT CAT TC T G A T C C C A G C T T
T A G T T G G A T GAAC T CG C A TG T CT CCA TAG A A T G C G C G C T AC T
T T C T T G A C A C C T T T T C G G C A T CG C C C T A A A A T T C G G C GT C
–35 region
Consensus
Pribnow box
TTGACA
TATA AT
Promoter sequence
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Figure 4.22
4.7 Transcription
• Consensus promoter sequence
• Pribnow Box and -35 region
• Core enzyme and holoenzyme
• Transcription start site
• Upstream and downstream
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4.7 Transcription
• Termination of RNA synthesis is governed by a
specific DNA sequence
• Intrinsic terminators: transcription is terminated
without any additional factors
• Rho-dependent termination: Rho protein recognizes
specific DNA sequences and causes a pause in the
RNA polymerase
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4.9 Transcription in Archaea and Eukarya
• The Archaea contain only a single RNA
polymerase
• Resembles eukaryotic polymerase II (Figure 4.21)
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Bacteria
Archaea
α
β
β'
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Eukarya
ω
Figure 4.21
4.9 Transcription in Archaea and Eukarya
• Archaea have a simplified version of eukaryotic
transcription apparatus
• Promoters and RNA polymerase similar to eukaryotes
(Figure 4.26)
• Regulation of transcription has major similarities with
Bacteria
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4.9 Transcription in Archaea and Eukarya
• Eukaryotic genes have coding and noncoding
regions
• Exons are the coding sequences
• Introns are the intervening sequences
• Are rare in Archaea
• Are found in tRNA and rRNA genes of Archaea
• Archaeal introns excised by special endonuclease
(Figure 4.27)
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4.9 Transcription in Archaea and Eukarya
• Eukaryotic RNA processing: many RNA
molecules are altered before they carry out their
role in the cell
• RNA splicing
• Takes place in nucleus
• Removes introns from RNA transcripts
• Performed by the spliceosome (Figure 4.28)
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4.9 Transcription in Archaea and Eukarya
• Eukaryotic RNA processing (cont'd)
• RNA capping (Figure 4.29)
• Addition of methylated guanine to 5′ end of mRNA
• Poly(A) tail (Figure 4.29)
• Addition of 100–200 adenylate residues
• Stabilizes mRNA and is required for translation
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4.11 Translation and the Genetic Code
• Transfer RNA aka tRNA acts as the adaptor
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4.11 Translation and the Genetic Code
Codon-anticodon interaction is base-pairing
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4.11 Translation and the Genetic Code
Wobble: irregular base
3′
5′
pairing allowed at third
Alanine tRNA
aka alanyl-tRNA
position of tRNA (Figure
4.32)
Anticodon
CGG
Key bases in codon:
anticodon pairing
5′
GC U
Wobble position;
base pairing more
flexible here
3′
mRNA
Codon
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Figure 4.32
© 2015 Pearson Education, Inc.
4.11 Translation and the Genetic Code
• Codon bias: multiple codons for the same amino
acid are not used equally
• Varies with organism
• Correlated with tRNA availability
• Cloned genes from one organism may not be translated
by recipient organism because of codon bias
• Some organelles and a few cells have slight
variations of the genetic code (e.g., mitochondria of
animals, Mycoplasma, and Paramecium)
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4.12 Transfer RNA
tRNA and amino acid brought together by aminoacyl-tRNA
3′
5′
synthetases
Acceptor stem
phe
3′ A
C
C
Acceptor
A
5′
C
G
end
Acceptor
G
C
stem
C
G
U
G
A
U
D loop
A
U
U
A
U
CC
G
AC AG
mA
U
A mG C U C A D
G
D
C T G U G U mC
CG AG A G
C
Ψ
G
U GA mG
G
mG
TΨC loop
G
C
G
C
Anticodon
U
A
mC G
stem
A
Y
A
mC
U
Y
A A mG
Anticodon
5′
U U C
Codon
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Acceptor
end
TΨC loop
D loop
Anticodon
stem
3′
mRNA
Anticodon loop
A
A
m
G
Anticodon
Figure 4.34
4.12 Transfer RNA
• Fidelity of recognition process between tRNA and
aminoacyl-tRNA synthetase is critical (Figure 4.35)
• The “Second Genetic Code”??????
• Incorrect amino acid could result in a faulty or
nonfunctioning protein
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5′ 3′
Amino acid
(valine)
tRNA acceptor stem
Uncharged
tRNA specific
for valine (tRNAVal)
AMP
Anticodon
region
Aminoacyl-tRNA
synthetase for valine
Linkage of valine
to tRNAVal
AMP
Valine
Charged valyl
tRNA, ready for
protein synthesis
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Anticodon
loop
Figure 4.35
4.13 Protein Synthesis
• Polysomes: a complex formed by ribosomes
simultaneously translating mRNA (Figure 4.37)
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4.14 Protein Folding and Secretion
• Once formed, a polypeptide folds to form a more
stable structure.
• Secondary structure
• Interactions of the R groups force the molecule to twist
and fold in a certain way (Figure 4.39)
• Tertiary structure
• Three-dimensional shape of polypeptide (Figure 4.40)
• Quaternary structure
• Number and types of polypeptides that make a protein
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A chain
α-Helix
B chain
Insulin
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Ribonuclease
β-Sheet
Figure 4.40
4.14 Protein Folding and Secretion
• Most polypeptides fold spontaneously into their
active form
• Some require assistance from molecular chaperones
or chaperonins for folding to occur (Figure 4.41)
• They only assist in the folding; they are not incorporated
into protein
• Can also aid in refolding partially denatured proteins
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ATP
Improperly
folded protein
“client” protein
ADP
DnaK
DnaJ
Properly folded
(active) protein
Transfer of
improperly
folded protein
to GroEL/ES
Molecular GroEL
chaperone
ATP
GroES
ADP
DnaK aka Hsp70
DnaJ aka Hsp40
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Properly folded
(active) protein
Figure 4.41
4.14 Protein Folding and Secretion
• Signal sequences: found on proteins requiring
transport from cell (Figure 4.42)
• 15–20 residues long
• Found at the beginning of the protein molecule
• Signal the cell's secretory system (Sec system)
• Prevent protein from completely folding
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Cytoplasmic
membrane
Translational
apparatus
Ribosome
SecA
Periplasm
Protein
Protein secreted
into periplasm
Proteins with
signal sequence
mRNA
Protein inserted
into membrane
Signal
recognition
particle
Protein does
not contain
signal sequence.
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Membrane
secretion
system
Figure 4.42
4.14 Protein Folding and Secretion
• Secretion of folded proteins: the Tat system
• Proteins that fold in the cytoplasm are exported by a
transport system distinct from Sec, called the Tat protein
export system
• Iron–sulfur proteins
• Redox proteins
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4.14 Protein Folding and Secretion
• Secretion of proteins: types I through VI (Figure
4.43)
• All are a large complex of proteins that form channels
through membranes
• Types II and V depend on Sec or Tat
• Types I, III, IV, and VI do not require Sec or Tat
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From gram-negative bacterial cell:
Cytoplasmic
membrane
Outer
membrane
The injectisome
traverses both the
cytoplasmic and
gram-negative
outer membranes.
Eukaryotic
cytoplasmic
membrane
Protein,
for example,
a toxin
Injectisome
(type III secretion
system) protein
complex
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Figure 4.43