6. Molecular Biology of Bacteria

8/10/2019 6. Molecular Biology of Bacteria

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Chapter 6

Molecular Biology of Bacteria


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OUTLINE

DNA Structure

Chromosome and Plasmids

DNA Replication Transcription

Translation

Protein Structures and Export


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Structure of Nucleotides

RNA DNA

Fig 6.1


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Fig 6.2

Hydrogen Bonding between Bases

GC paring is

stronger than AT

pairing


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DNA Has Major and Minor Grooves

120

240

Base pairing angle

relative to backbone

creates major and minor

grooves in the 3-D DNA

structure.


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Helical Structure of DNA


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Fig 6.7

Thermal

Denaturation ofDNA

Single-stranded DNA

has higher absorbance

at 260 nm


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Supercoiling is required

E. coli cell size: 12 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.

DNA Supercoiling


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DNA Gyrase (topoisomerase II)

Creates Negative Supercoiling in DNA

Fig 6.9


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Chromosome and Plasmid


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Escherichia coli Chromosome

Circular

4.64 Mbp = 4,640 kbp = 4,639,221 bp 4288 protein-encoding genes (88% of the chromosome)

Genetic map ofE. coliis 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 inE. 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 theE. coli genome originated from horizontal transfer

(distinct GC ratio, codon distributions; pathogenicity islands)


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Fig 6.10

Escherichia coli MG1655 Chromosome


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Plasmids

Genetic elements that replicate independently of the host

chromosome, so plasmids should carry genes for their ownreplication.

Typical plasmids are circular double strand DNA molecules

with the size of 3-10 kbp.

Plasmid incompatibility: two closely related plasmids cannotbe 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 tragenes


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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

BacteriocinsColicins (E. coli), Pesticins (Yersinia pestis), Nisin A

(Lactic acid bacteria)

Metabolic function


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Fig 6.12

Genetic Map of R100

R100 provides resistanceto several antibiotics and

metal:

cat: chloramphenicolstr: streptomycin

sul: sulfonamides

tet: tetracycline

mer: mercury

Found in enteric bacteria


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Functions of Plasmids


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Replication

Transcription

Translation

DNA polymerase

RNA polymerase

Ribosome

Central Dogma


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DNA Replication


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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 atthe 3end.

resulting in asymmetric leading and lagging strands.

Two Important Characteristics of

(all) DNA Polymerases


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Five DNA Polymerases inE. coli

error-prone


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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 35 exonuclease activity

Processivity:

Ability to perform multiple catalytic cycles without

dissociating with the template.

Clamp


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Fig 6.16

Events at the Replication Fork


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Fig 6.18

Joining Okazaki

Fragmentsin the Lagging Strand


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Bidirectional

ReplicationDNA synthesis is

bidirectional in

prokaryotes

Fig 6.20

Fig 6.19


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Q.It takes 40 min to replicate the wholeE.

coli chromosome. However, under the best

condition,E. coligrow with a doubling time of

20 min. then what is the solution?

Multiple DNA replication forks


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Fig 6.21

Multiple DNA Replication Forks

DNA Replication and Cell Division


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DNA Replication Is Very Accurate

Mutational rate is 10-810-11

Two mechanisms of fidelity

Correct base insertion filter by the active site

Proofreading (35 exonuclease activity)


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Q. Mutation rates ofE. colifrom DNA replication

are 1081011errors per base inserted. After

complete DNA replication (of the chromosome),

how many point mutation(s) do you expect out of

copying?

A)


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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?

PCR (Polymerase Chain Reaction) Is

Essentially DNA Replication in vitro


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Transcription


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Bacterial RNA Polymerase Consists of

Multiple Subunits

2 +

Crab claw shape of

RNA polymerase

Si F t R i


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2012 Pearson Education, Inc.Fig 6.26

Sigma Factor Recognizes

Where to Start Transcription

A promoteris

composed of two

important sequences:

-35 sequence

-10 sequence

If a promoter is closeto a consensus

sequence, the

promoter is strong

l i i i iff


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Alternative Sigma Factors Recognize Different

Sequences and Serve Specific Roles


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Transcription Elongation

h d d


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Fig 6.27

Rho-Independent

Transcription Termination

DNA-RNA interaction is

significantly diminished

because of the self

complementary stem-

loop structure and the

weakest A-U interactions


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Comparison of Rho-Independent and

Rho-Dependent Termination


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Translation


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tRNA Is the Information Bridge

Fig 6.33

Amino acid information

Codon information


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tRNA Structure

Fig 6.33cloverleaf representation 3D model


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Fig 6.34

Aminoacyl-tRNA Synthetase

Amino acid + ATP aminoacyl-AMP + PPi

Aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP


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Fig 6.35

Protein Synthesis Steps InitiationmRNA binds small ribosome

subunit

Elongation

Requires the elongation

factors of EF-Tu and EF-Ts

TranslocationRequries the elongation

factor of EF-G

Termination

Release factors recognize

stop codons and cleave the

attached polypeptide from

the final tRNA


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Codon

recognition

Peptide bond

formationTranslocation


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Q.If a ribosome reaches the end of an

mRNA molecule and there is no stop

codon, what will happen?

i d ( ll d) ib


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Freeing Trapped (Stalled) Ribosomes

tmRNAacts 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

R l f Rib l RNA


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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


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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

incorporatingN-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


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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'

Genetic Code

I ti f


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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 sequencedownstream of each stop

codon encoding the amino

acid


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Protein Structures

and Export

l f i S


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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

S d S f P l id


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Secondary Structure of Polypeptides

Ch i A i P i F ldi


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Chaperonins Assist Protein Folding

Chaperonins = molecular chaperones

Functions

Folding newly synthesized proteins(keep them from folding too abruptly)

Refolding proteins that have partially denatured

F K Ch i i E li


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Fig 6.40

Four Key Chaperonins inE. coli

Protein Export and Secretion


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Protein Export and Secretion

Protein Export: CytoplasmPeriplasm

Protein Secretion: CytoplasmOutside 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

SecAor SRP(signal recognition particle).

Some proteins must be exported in a fully folded state

(because they cannot be folded otherwise) by the Tat

system.

E t f t i


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Export of proteins

via the Major Secretory System

6. Molecular Biology of Bacteria

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