1. I. The Blueprint of Life: Structure of the Bacterial Genome
4.3 Genetic Elements: Chromosomes and Plasmids

2. 4.3 Genetic Elements: Chromosomes and Plasmids
Genome: entire complement of genes in cell or in a particular genetic element
Chromosome: main genetic element in prokaryotes, usually a single piece of DNA
Other genetic elements include virus genomes, plasmids, transposable elements (jumping genes) and genomes of eukaryotic organelles

3. 4.3 Genetic Elements: Chromosomes and Plasmids
Viruses are subcellular parasitic genetic elements
Extracellular phase
May be either RNA or DNA
Called “phages”

4. 4.3 Genetic Elements: Chromosomes and Plasmids
Plasmids: DNA that replicates separately from chromosome
“autonomous”
“episome”
Great majority are double-stranded
Most are circular
Generally beneficial for the cell (e.g., antibiotic resistance)
NOT extracellular, unlike viruses

5. 4.3 Genetic Elements: Chromosomes and Plasmids
A plasmid is an accessory genetic element that is expendable and rarely contains genes for growth under all conditions-extra genes
Contrasts with chromosome which is the main genetic element and has essential or with "housekeeping" genes

6. 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
Main types:
Insertion sequences (IS)
Transposons (Tn)
Certain viruses

7. 4.3 Genetic Elements: Chromosomes and Plasmids
The Escherichia coli chromosome- a well understood example
The E. coli chromosome from strain K-12 MG1655 has been mapped using a combination of biological and biochemical methods (Figure 4.8)

8. Figure 1 The overall structure of theE. coli genome.
The overall structure of theE. coli genome. The origin and terminus of replication are shown as green lines, with blue arrows indicating replichores 1 and 2. A scale indicates the coordinates both in base pairs and in minutes (actually centisomes, or 100 equal intervals of the DNA). The distribution of genes is depicted on two outer rings: The orange boxes are genes located on the presented strand, and the yellow boxes are genes on the opposite strand. Red arrows show the location and direction of transcription of rRNA genes, and tRNA genes are shown as green arrows. The next circle illustrates the positions of REP sequences around the genome as radial tick marks. The central orange sunburst is a histogram of inverse CAI (1 – CAI), in which long yellow rays represent clusters of low (<0.25) CAI. The CAI plot is enclosed by a ring indicating similarities between previously described bacteriophage proteins and the proteins encoded by the complete E. coli genome; the similarity is plotted as described in Fig. 3 for the complete genome comparisons.
Frederick R. Blattner et al. Science 1997;277:
Published by AAAS

9. Figure 4.8 The chromosome of Escherichia coli strain K-12.

10. 4.3 Genetic Elements: Chromosomes and Plasmids
Some features of the E. coli chromosome
4,639,221 bp = 4.63 Mbp
4288 protein-coding genes
38% unknown function
93 RNA coding genes (rRNA and tRNA)
Genome contains jumping genes and viral genes

11. 4.3 Genetic Elements: Chromosomes and Plasmids
Shows Genetic Economy
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
Average protein contains ~300 amino acids
Genes are close together
Few repeated genes
Few intervening sequences or introns

12. 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
Name begins with “p” e.g. pBGH7
Abundance (copy number) is variable: relaxed vs stringent

13. Figure 4.9 The bacterial chromosome and bacterial plasmids, as seen in the electron microscope.

14. 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

15. 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

16. Figure 4.10 Genetic map of the resistance plasmid R100.

18. 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

19. 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

20. 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

21. Gene(s) to be transcribed
RNA
polymerase
(core
enzyme)
Sigma
factor
Sigma recognizes
promoter and
initiation site. 5′ 3′ 3′ 5′
Promoter region
Gene(s) to be transcribed
(light green strand)
Transcription begins;
sigma released. RNA
chain grows.
Sigma 5′ 3′ 3′ 5′ 5′
RNA 5′ 3′ 3′ 5′
Termination site
reached; chain
growth stops. 5′ 5′ 3′ 3′ 5′ 5′
Polymerase and RNA
released.
Figure 4.20 Transcription. 5′ 3′ 3′ 5′ 3′ 5′
DNA
Short transcripts
Longer transcripts
Figure 4.20

22. Figure 4.22 RNA polymerase (core enzyme) Transcription 5′ 3′ 3′ 5′
Sigma
mRNA start 5′ 3′ 1. C T T G A G A T C T T G C A C G C T A G A G T A T G C T G A C T G A A C G T A T G T A C T A C G A C T G T C A G C A T C T G A A G C T A T C A G T T G C A T C G T A C T T A C A G T A G C G A T A T C G G A C T G C A C G G A C G A C G A T A C T A C G T 2.
Figure 4.22 The interaction of RNA polymerase with a bacterial promoter. 3. 4. 5. 6.
–35 region
Pribnow box
Consensus T T G A C A T A T A A T
Promoter sequence
Figure 4.22

23. 4.7 Transcription Consensus promoter sequence
Pribnow Box and -35 region
Core enzyme and holoenzyme
Transcription start site
Upstream and downstream

24. 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

25. 4.11 Translation and the Genetic Code
Transfer RNA aka tRNA acts as the adaptor

26. 4.11 Translation and the Genetic Code
Codon-anticodon interaction is base-pairing

27. 4.11 Translation and the Genetic Code3′
Wobble: irregular base pairing allowed at third position of tRNA (Figure 4.32) 5′
Alanine
tRNA
Anticodon C G G
Wobble position;
base pairing more
flexible here
Key bases in codon:
anticodon pairing
Figure 4.32 The wobble concept. 5′ G C U 3′
mRNA
Codon
Figure 4.32

29. 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)

30. 4.12 Transfer RNA tRNA and amino acid brought together by aminoacyl-tRNA synthetases
Acceptor stem 5′ 3′
phe 3′ A
Acceptor
end C
TΨC loop C
Acceptor end A 5′ C G
Acceptor stem G C C G U G U A A U
D loop U C A U
D loop mA C G A C A G U A A mG C U C D G D C G U G U mC T C C G A G G
Anticodon
stem Ψ A U G G
TΨC loop mG A mG G G C G C U A
Anticodon stem mC G
Figure 4.34 Structure of a transfer RNA. Y A A mC Y U A A A mG 5′
Anticodon A
Anticodon 3′ mG U U C
mRNA
Anticodon loop
Codon
Figure 4.34

31. 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

32. Figure 4.35 Figure 4.35 Aminoacyl-tRNA synthetase. 5′ 3′ Amino acid
(valine)
tRNA acceptor stem
Uncharged
tRNA specific
for valine (tRNAVal)
AMP C A
Anticodon
region
Aminoacyl-tRNA
synthetase for valine
Linkage of valine
to tRNAVal
AMP
Figure 4.35 Aminoacyl-tRNA synthetase.
Valine
Charged valyl
tRNA, ready for
protein synthesis
Anticodon
loop C A
Figure 4.35

33. 4.13 Protein Synthesis Two key points
Polysomes: a complex formed by ribosomes simultaneously translating mRNA (Figure 4.37)
tmRNA: a small RNA molecule that rescues stalled ribosomes

34. 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

35. DnaK aka Hsp70 DnaJ aka Hsp40 Figure 4.41 ATP ATP ADP
Improperly folded protein
“client” protein
DnaK
DnaJ
Properly folded
(active) protein
Transfer of
improperly
folded protein
to GroEL/ES
GroEL
ATP
Molecular
chaperone
ADP
GroES
Figure 4.41 The activity of molecular chaperones.
DnaK aka Hsp70
DnaJ aka Hsp40
Properly folded
(active) protein
Figure 4.41

36. 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

37. Figure 4.42 Cytoplasmic membrane SecA Periplasm Protein
Translational
apparatus
SecA
Periplasm
Protein
Protein secreted
into periplasm
Ribosome
Proteins with
signal sequence
mRNA
Protein inserted
into membrane
Figure 4.42 Export of proteins via the major secretory system.
Signal
recognition
particle
Protein does
not contain
signal sequence.
Membrane
secretion
system
Figure 4.42

38. 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

39. 4.14 Protein Folding and Secretion
Secretion of proteins: Types I through VI systems (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

40. From gram-negative bacterial cell:
The injectisome is a special type of Sec III System
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
Figure 4.43 Secretion of molecules in gram-negative bacteria using the type III "injectisome" system.
Figure 4.43