Chapter 6

Molecular Biology of Bacteria

OUTLINE

DNA Structure
Chromosome and Plasmids
DNA Replication
Transcription
Translation
Protein Structures and Export

Structure of Nucleotides

RNA

DNA
Fig 6.1

Hydrogen Bonding between Bases

GC paring is
stronger than AT
pairing

Fig 6.2

DNA Has Major and Minor Grooves

240
120
Base pairing angle
relative to backbone
creates major and minor
grooves in the 3-D DNA
structure.

Helical Structure of DNA

Thermal
Denaturation of
DNA
Single-stranded DNA
has higher absorbance
at 260 nm

Fig 6.7

DNA Supercoiling
Supercoiling is required
E. coli cell size: 1 2 mm
E. coli chromosome size: 1 mm

DNA can be overwound

(positive supercoiling) or
underwound (negative
supercoiling).

Negative supercoiled DNA

is the form predominantly
found in nature.

Chromosomal DNA Contains

Many Supercoiled Domains

A nick in the DNA of

one domain does not
relax DNA in the other
domains, so damage
can be localized

Fig 6.8

DNA Gyrase (topoisomerase II)

Creates Negative Supercoiling in DNA

Fig 6.9

Chromosome and Plasmid

Escherichia coli Chromosome

Circular
4.64 Mbp = 4,640 kbp = 4,639,221 bp
4288 protein-encoding genes (88% of the chromosome)
Genetic map of E. coli is in minutes.
Genes are clustered into operons, but operons are NOT the rule in
E. coli (70% of the transcriptional units contain a single gene).
Many genes that are highly expressed in E. coli are oriented so
that they are transcribed in the same direction that the DNA
replication fork moves through them.
Many of the protein-encoding genes arose by gene duplication
during evolutionary history
20% of the E. coli genome originated from horizontal transfer
(distinct GC ratio, codon distributions; pathogenicity islands)

Escherichia coli MG1655 Chromosome

Fig 6.10

Plasmids

Genetic elements that replicate independently of the host

chromosome, so plasmids should carry genes for their own
replication.
Typical plasmids are circular double strand DNA molecules
with the size of 3-10 kbp.
Plasmid incompatibility: two closely related plasmids cannot
be maintained in the same cell at the same time.
Inc (incompatibility) groups
Plasmids can be diluted out from host cells (called curing)
because they are not essential.
Plasmids can be transferred to bacteria via conjugation (and
less effectively transformation)
Transfer requires a set of tra genes

Plasmids Are Not Essential,

but Provide Usefulness to Host Cells
Resistance (R plasmids)
Confer resistance to antibiotics
e.g.) R100

Virulence
Attachment/colonization function
production of virulence factors

Bacteriocins
Colicins (E. coli), Pesticins (Yersinia pestis), Nisin A
(Lactic acid bacteria)

Metabolic function

Genetic Map of R100

R100 provides resistance
to several antibiotics and
metal:

cat: chloramphenicol
str: streptomycin
sul: sulfonamides
tet: tetracycline
mer: mercury
Found in enteric bacteria
Fig 6.12

Functions of Plasmids

Central Dogma
Replication

DNA polymerase

Transcription

RNA polymerase

Translation

Ribosome

DNA Replication

Two Important Characteristics of

(all) DNA Polymerases
1. they can only extend nucleic acid chains:
i.e., they cannot initiate new ones.
absolutely requires a primer (made by primase).
2. they add mononucleotides to the 3 hydroxyl of
deoxyribose and therefore elongate nucleic acid only at
the 3end.
resulting in asymmetric leading and lagging strands.

Five DNA Polymerases in E. coli

error-prone

Other Functions Are Required for

DNA Replication (Replisome)
Strand separation:
Separate and maintain single-stranded DNA (helicase
and single-strand binding protein)
Handle supercoiling (DNA gyrase)
Fidelity:
Ability to put correct bases.
Proofreading via 3 5 exonuclease activity
Processivity:
Ability to perform multiple catalytic cycles without
dissociating with the template.
Clamp

Events at the Replication Fork

Fig 6.16

Joining Okazaki
Fragments
in the Lagging Strand

Fig 6.18

Bidirectional
Replication
DNA synthesis is
bidirectional in
prokaryotes
Fig 6.19

Fig 6.20

Q. It takes 40 min to replicate the whole E.

coli chromosome. However, under the best
condition, E. coli grow with a doubling time of
20 min. then what is the solution?
Multiple DNA replication forks

DNA Replication and Cell Division

Fig 6.21

Multiple DNA Replication Forks

DNA Replication Is Very Accurate

Mutational rate is 10-8 10-11
Two mechanisms of fidelity
Correct base insertion filter by the active site
Proofreading (3 5 exonuclease activity)

Q. Mutation rates of E. coli from DNA replication

are 1081011 errors per base inserted. After
complete DNA replication (of the chromosome),
how many point mutation(s) do you expect out of
copying?
A) <1
B) 1
C) ~10
D) ~100

PCR (Polymerase Chain Reaction) Is

Essentially DNA Replication in vitro
1. What dissociate double-stranded DNA?
2. Do we use RNA primer or DNA primer for
PCR?
3. DNA polymerase, what might be the most
critical property for the polymerase that is
used for PCR?
4. Per every PCR cycle, what is the maximum
fold-increase in DNA copy?

Transcription

Bacterial RNA Polymerase Consists of

Multiple Subunits

2w +

Crab claw shape of

RNA polymerase

Sigma Factor Recognizes

Where to Start Transcription
A promoter is
composed of two
important sequences:
-35 sequence
-10 sequence
If a promoter is close
to a consensus
sequence, the
promoter is strong
2012 Pearson Education, Inc.

Fig 6.26

Alternative Sigma Factors Recognize Different

Sequences and Serve Specific Roles

Transcription Elongation

Rho-Independent
Transcription Termination

Fig 6.27

DNA-RNA interaction is
significantly diminished
because of the self
complementary stemloop structure and the
weakest A-U interactions

Comparison of Rho-Independent and

Rho-Dependent Termination

Translation

tRNA Is the Information Bridge

Amino acid information

Codon information

Fig 6.33

tRNA Structure

cloverleaf representation

Fig 6.33

3D model

Aminoacyl-tRNA Synthetase
Amino acid + ATP
Aminoacyl-AMP + tRNA

aminoacyl-AMP + PPi
aminoacyl-tRNA + AMP

Fig 6.34

Protein Synthesis Steps

Initiation
mRNA binds small ribosome
subunit

Elongation
Requires the elongation
factors of EF-Tu and EF-Ts

Translocation
Requries the elongation
factor of EF-G

Termination

Fig 6.35

Release factors recognize

stop codons and cleave the
attached polypeptide from
the final tRNA

Codon
recognition

Peptide bond
formation

Translocation

Q. If a ribosome reaches the end of an

mRNA molecule and there is no stop
codon, what will happen?

Freeing Trapped (Stalled) Ribosomes

tmRNA acts as both
tRNA (carrying alanine)
and mRNA that contains
(i) codons for a peptide
(susceptible to protease)
and (ii) a stop codon
(recruiting release factor).

Fig 6.37

Role of Ribosomal RNA

in Protein Synthesis
16S rRNA: base pairing with the Shine-Dalgarno
sequence (initiation).
23S rRNA: peptidyl transferase activity
Other ribosomal RNA functions:
Positioning tRNA in the A and P sites
Ribosome subunit dissociation
Translocation

Genetic Code, Codons and Codon Bias

Codons are degenerate (redundant)
64 (444) codons for 20 amino acids.
One lysyl tRNA can bind to both AAA and AAG codons
(Wobble).
There are three stop codons (UAA, UAG and UGA).
AUG (sometimes GUG or UUG) is the start codon
incorporating N-formylmethionine.
In an organism, some codons are greatly preferred over
others even though they encode the same amino acid
(codon bias).
The genetic code is universal, but there are slight
variations: e.g. UGA to encode tryptophan.

Genetic Code
Q. Under the condition where methionine must be the first
amino acid, what is the third amino acid of the protein
encoded by the following mRNA?
5'-CCUCAUAUGCGCCAUUAUAAGUGACACACA-3'

Incorporation of
Selenocysteine and Pyrrolysine
Both amino acids are rare.
Both are encoded by stop
codons (UGA and UAG,
respectively)
Both have specific
aminoacyl tRNA transferase
Incorporation of both rely
on a recognition sequence
downstream of each stop
codon encoding the amino
acid

Protein Structures
and Export

Levels of Protein Structure

Primary structure
Amino acid sequence
Secondary structure
Depends largely on hydrogen bonding
a-helix
b-sheet
Tertiary structure
Depends largely on hydrophobic interaction
Quaternary structure
Multiple subunits

Secondary Structure of Polypeptides

Chaperonins Assist Protein Folding

Chaperonins = molecular chaperones
Functions
Folding newly synthesized proteins
(keep them from folding too abruptly)
Refolding proteins that have partially denatured

Four Key Chaperonins in E. coli

Fig 6.40

Protein Export and Secretion

Protein Export: Cytoplasm Periplasm
Protein Secretion: Cytoplasm Outside of the cell

Signal sequence (15-20 amino acids) is required for

cell membrane, periplasmic and secreted proteins.
Most proteins are exported in an unfolded state by
SecA or SRP (signal recognition particle).
Some proteins must be exported in a fully folded state
(because they cannot be folded otherwise) by the Tat
system.

Export of proteins
via the Major Secretory System

Fig 6.41