Module 6 Microbial Genetics - University of Hawaii · 2014. 10. 20. · Genetics: science of heredity ! Study of what genes are, how they determine the characteristics of an organism,

© 2013 Pearson Education, Inc.

Module 6 Microbial Genetics

Copyright © 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case

Chapter 8

Microbial Genetics

© 2013 Pearson Education, Inc. Lectures prepared by Helmut Kae


Structure and Function of the Genetic Material §  Genetics: science of heredity

§  Study of what genes are, how they determine the characteristics of an organism, how they carry information, how the information is copied, how information is passed on to subsequent generations and between organisms

§  Genome: all the genetic information in a cell §  Includes chromosomes and plasmids

§  Genomics: sequencing, characterization of genomes


Structure and Function of the Genetic Material §  Chromosomes: a structure that contains the DNA

§  Physically carries the hereditary information, genes §  Bacteria typically have one circular chromosome

DNA is twisted and supercoiled to fit into cell

Chromosome is 1000x longer than width of cell

Chromosome erupting from one E. coli cell


Structure and Function of the Genetic Material §  Genes: segments of DNA that carry information to

produce functional products, proteins §  Genetic code: the set of rules that determines how

a nucleotide sequence of a gene is converted into the amino acid sequence of a protein §  Linear sequence of nucleotides, bases provides the

information for making proteins §  Much of anabolism is making proteins from DNA

§  When a product is made, the gene is expressed


Structure and Function of the Genetic Material §  Genotype: an organism’s genetic makeup

§  The information found in its DNA §  Represents potential characteristics

§  Phenotype: an organism’s expressed properties §  Eg, an ability to resist an antibiotic

§  Phenotype is the display of an organism’s genotype §  Genotype is collection of DNA in cell §  Phenotype is collection of proteins in cell


Insert Fig 8.2

Parent cell

DNA

expression

Genetic information is used within a cell to produce the proteins needed for the cell to function.

Transcription

Translation

recombination

Genetic information can be transferred between cells of the same generation.

New combinations of genes

Cell metabolizes and grows Recombinant cell Daughter cells

replication

Genetic information can be transferred between generations of cells.

Figure 8.2 The Flow of Genetic Information.

Genetic information can flow in a number of ways

“Horizontal gene transfer”

“Vertical gene transfer”


DNA

§  Polymer of nucleotides: adenine, thymine, cytosine, and guanine

§  Strands are held together by hydrogen bonds §  AT and CG

§  Strands are antiparallel §  DNA has direction - 3’ (3 prime) and 5’ (5 prime) - DNA strands are counter-parallel to each other


Insert Fig 8.3b

The two strands of DNA are antiparallel. The sugar-phosphate backbone of one strand is upside down relative to the backbone of the other strand. Turn the book upside down to demonstrate this.

3′ end

5′ end

5′ end

3′ end

Figure 8.3b DNA replication.


DNA

§  One parental DNA molecule converted into 2 identical daughter molecule §  Parental DNA strand acts as template for new strand


Insert Fig 8.3a

1

Daughter strand

3

2

1

Replication fork

3′ end Parental strand

5′ end Parental strand Daughter

strand forming

Parental strand Parental

strand

3′ end 5′ end Figure 8.3a DNA replication.

Double stranded DNA unwound by helicase enzyme

Exposed bases matched up with bases in cytoplasm

A to T C to G

DNA polymerase joins new nucleotides to

forming DNA molecule

Each new DNA molecule contains one parental strand, one daughter strand à semi-conservative replication


Insert Fig 8.2

Parent cell

DNA

expression


Transcription

Translation

recombination




replication







RNA and Protein Synthesis

§  Genetic information from DNA follows the “Central Dogma of Biology” §  DNA is used to make RNA, which is used to make protein §  DNA à RNA à Protein

§  DNA à RNA: Transcription, RNA synthesis §  RNA à Protein: Translation, Protein synthesis


Transcription

§  Synthesis of RNA §  Using DNA as a template

§  Recall, RNA is single stranded, uses U instead of T §  Three kinds of RNA

§  Ribosomal RNA, rRNA: integral part of ribosomes §  Transfer RNA, tRNA: involved in protein synthesis §  Messenger RNA, mRNA: carries information for making

protein §  mRNA is synthesized from a gene by enzyme called

RNA polymerase


Transcription

§  Transcription begins when RNA polymerase binds to the promoter sequence

§  RNA polymerase joins nucleotides into new mRNA strand using DNA as template §  New mRNA complementary to DNA template §  A à U §  T à A §  C à G §  G à C


Insert Fig 8.7

RNA polymerase

DNA RNA polymerase binds to the

promoter, unwinds DNA RNA is synthesized by complementary base pairing with the nucleotides on the template strand of DNA.

Transcription continues until RNA Polymerase

reaches the terminator.

RNA and RNA polymerase are released, and the DNA helix re-forms.

Promoter (gene begins)

RNA polymerase Template strand of DNA

RNA RNA nucleotides

RNA synthesis

Terminator (gene ends)

Figure 8.7 The process of transcription.

DNA template is read 3’ à 5’ mRNA is made 5’ à 3’


Transcription

§  RNA is complementary to DNA §  If DNA template is CGATAAG

§  What is the RNA strand formed?

§  RNA synthesized is GCUAUUC


Insert Fig 8.2

Parent cell

DNA

expression


Transcription

Translation

recombination




replication







Translation

§  “Translates” the “language” of nucleic acids into “language” of proteins

§  Codons: groups of three bases used to translate nucleic acids into amino acids §  Each codon “codes” for an amino acid §  Sequence of codons on mRNA determines sequence of

amino acids §  Codons to amino acids is the genetic code


The Genetic Code

§  Written as mRNA, 5’ to 3’ §  Two types of codons §  Sense codons: code for

amino acids §  61 codons for 20 amino acids §  Degeneracy of genetic code –

amino acids coded for by multiple codons

§  Nonsense codons: code for stops in translation §  Aka stop codons


Translation

§  Translation starts with AUG start codon §  Codes for amino acid methionine §  In Bacteria, translation starts with formylmethionine

§  tRNA carries amino acids to ribosomes

tRNA carries amino acid on one end …

… and has anticodon at other end Anticodon recognizes codon on

mRNA What’s the codon this tRNA recognizes?


1

TRANSLATION

DNA

mRNA

Protein Ribosomal subunit

tRNA

Anticodon

Ribosomal subunit

Met

1 Components needed to begin translation come together.

mRNA

Figure 8.9.1-2 The process of translation.


RNA polymerase

DNA Met Leu

mRNA


Ribosome binds at start codon

tRNA binds to next codon


1

DNA

Met

Met Met

Met

Peptide bond forms

Leu Leu Gly

Phe

mRNA

A site E site

mRNA Ribosome moves along mRNA


Ribosome forms peptide bond between

amino acids

Ribosome moves along mRNA in 5’

to 3’ direction


Insert Fig 8.9 (5) and (6)

tRNA released Met Met

Leu

Leu Growing polypeptide chain

Gly Phe

Gly

Phe Met

mRNA

mRNA

5 The second amino acid joins to the third by another peptide bond, and the first tRNA is released from the E site.

6 The ribosome continues to move along the mRNA, and new amino acids are added to the polypeptide.



Insert Fig 8.9 (7) and (8)

Met Met

Met Met Polypeptide released

Arg Met

Gly Met

Phe

Gly

mRNA

Stop codon

New protein

mRNA

Gly

Met Leu

Leu Phe Met

Arg


Translation continues until ribosome

reaches stop codon

Ribosome breaks up, mRNA, protein

released


Transcription and Translation

Consider the following "Template" strand of DNA AGA GGA TTA TAC TAT TCG TGG CGC AGT ATG TAA ACT TCT 1.  Transcribe this DNA molecule 2.  Translate the mRNA to find the "hidden message" in the protein.

a.  Hint #1 - Start at the first codon, not the first AUG b.  Hint #2 - Use the first letter of each amino acid to spell out the

message


AGA GGA TTA TAC TAT TCG TGG CGC AGT ATG TAA ACT TCT


Mutation

§  A change in the genetic material §  Mutations may be neutral, beneficial, or harmful §  Mutagen: agent that causes mutations

§  Induced mutations §  Spontaneous mutations: occur in the absence of

a mutagen


Types of Mutations

§  Base substitution (point mutation): one base is replaced by a different base §  May cause change in one amino acid, create a stop codon

§  Frameshift mutation: one or a few bases are deleted or inserted (not in multiples of three) §  Shifts “translational reading frame” of mRNA §  Causes change in all amino acids after frameshift §  Almost always results in nonfunctional protein


Insert Fig 8.17

During DNA replication, a thymine is incorporated opposite guanine by mistake.

Parental DNA

Replication

Daughter DNA

Daughter DNA In the next round of replication, adenine pairs with the new thymine, yielding an AT pair in place of the original GC pair.

Daughter DNA

Replication

Granddaughter DNA

When mRNA is transcribed from the DNA containing this substitution, a codon is produced that, during translation, encodes a different amino acid: tyrosine instead of cysteine.

Transcription

mRNA

Amino acids Cysteine Tyrosine

Translation

Cysteine Cysteine

Figure 8.17 Base substitutions.


Translation

DNA (template strand)

Transcription

mRNA

Amino acid sequence Met Lys Phe Gly Stop

Normal DNA molecule

Figure 8.18ad Types of mutations and their effects on the amino acid sequences of proteins.

Frameshift mutation

Met Lys Leu Ala


Outcomes of Mutation

§  Silent mutations: mutations that have no effect §  Change in base, no change in amino acid §  Due to degeneracy of genetic code

§  Missense mutations: mutations that result in an amino acid substitution in protein

§  Nonsense mutations: mutation that introduces premature stop codon


Translation


Transcription

mRNA


Normal DNA molecule

Figure 8.18a-b Types of mutations and their effects on the amino acid sequences of proteins.


Amino acid sequence Lys Phe Ser

mRNA

Met Stop

Missense mutation


Translation


Transcription

mRNA


Normal DNA molecule

Figure 8.18ac Types of mutations and their effects on the amino acid sequences of proteins.

Nonsense mutation

Met Stop


§  Spontaneous mutation rate = 1 in 109 replicated base pairs or 1 in 106 replicated genes

§  Mutagens increase to 10–5 or 10–3 per replicated gene

The Frequency of Mutation


Chemicals Mutagens

§  Interact with DNA to cause improper base pairing, deletions, insertions

HNO2 alters A

“A” binds with C instead of T

Analogs of DNA look like bases

But don’t base pair properly


Radiation Mutagens

§  Ionizing radiation – X-rays, gamma rays §  Ionize molecules §  Cause structural damage to DNA, leads to errors in DNA

replication §  Nonionizing radiation – UV light

§  Cause deletions of adjacent thymines


Ultraviolet light

Thymine dimer

New DNA

Exposure to ultraviolet light causes adjacent thymines to become cross-linked, forming a thymine dimer and disrupting their normal base pairing.

An endonuclease cuts the DNA, and an exonuclease removes the damaged DNA.

DNA polymerase fills the gap by synthesizing new DNA, using the intact strand as a template.

DNA ligase seals the remaining gap by joining the old and new DNA.

Figure 8.21 The creation and repair of a thymine dimer caused by ultraviolet light.


Insert Fig 8.2

Parent cell

DNA

expression


Transcription

Translation

recombination




replication







Genetic Recombination

§  Exchange of genes between 2 DNA molecules §  Contributes to genetic diversity of population

§  In eukaryotes, genetic recombination happens regularly as part of sexual cycle §  Recombination within one organism

§  In prokaryotes, transfer of genes happens by: §  Vertical gene transfer

§  Horizontal gene transfer (recombination between 2

organisms)


Horizontal Gene Transfer

§  Donor transfers part of its genome to recipient cell §  Recipient can incorporate (recombine) part of donor

DNA §  Rest is degraded for use as building blocks

§  Recipient that incorporates DNA is called recombinant

§  Rare event, occurs in less than 1% of population §  Three mechanisms of horizontal gene transfer

§  Transformation, conjugation, transduction


Transformation Transfer of “naked” DNA

“Naked” DNA in environment

Recipient cell

Transformation - uptake of

DNA Recombination – integration of

DNA into genome

Cell that recombines foreign DNA is a recombinant cell


RECOMBINATION

Mouse died. Mouse died. Mouse remained healthy. Mouse remained healthy.

Colonies of encapsulated bacteria were isolated from dead mouse.

Colonies of encapsulated bacteria were isolated from dead mouse.

No colonies were isolated from mouse.

A few colonies of non-encapsulated bacteria were isolated from mouse; phagocytes destroyed nonencapsulated bacteria.

Living encapsulated bacteria injected into mouse.

Living nonencapsulated bacteria injected into mouse.

Heat-killed encapsulated bacteria injected into mouse.

Living nonencapsulated and heat-killed encapsulated bacteria injected into mouse.

Figure 8.25 Griffith’s experiment demonstrating genetic transformation.


Transformation

§  In nature, some bacteria release DNA into environment §  After cell death, cell lysis

§  Some bacteria can take up this DNA via transformation §  Occurs naturally in Bacillus, Haemophilus, Neisseria,

Acinetobacter §  Competence: physiological state in which recipient

cell can take up DNA via transformation §  ie, Haemophilus can take up DNA only when competent


Transduction

§  DNA transferred as a part of bacteriophage infection §  Two types of transduction

§  Generalized transduction - phage mediated transfer of random segments of DNA

§  Specialized transduction - phage mediated transfer of specific genes - ie, some phages transfer only toxin genes


Insert Fig 8.29

RECOMBINATION

Many cell divisions

Recombinant cell reproduces normally

Donor bacterial DNA

Recipient bacterial DNA

Bacterial DNA

Phage DNA

Donor cell

Phage protein coat

Phage DNA

Bacterial chromosome

Recipient cell

A phage infects the donor bacterial cell. Injects phage DNA.

Phage DNA and proteins are made, and the bacterial chromosome is broken into pieces.

Phage particles assembled. Some phages mistakenly package bacterial DNA

A phage carrying bacterial DNA (transducing phage) infects a new host cell, the recipient cell.

Recombination can occur, producing a recombinant cell with a genotype different from both the donor and recipient cells.

5

Figure 8.29 Transduction by a bacteriophage.



Plasmids

§  Plasmids are self-replicating circular molecules of DNA §  Small, about 1-5% of genome §  Often carry genes that are not essential for survival

§  Conjugative plasmids: carry genes for plasmid transfer §  Conjugation


Conjugation

§  Plasmid dependent DNA transfer §  Requires cell to cell contact §  Conjugating cells must be opposite “mating types”

§  Donor cell must carry plasmid §  Recipient cell must not carry plasmid §  “Bacterial sex”


Insert Fig 8.27

Sex pilus Mating bridge

Sex pilus

F+ cell

F– cell

Mating bridge

Figure 8.27 Bacterial conjugation.


Insert Fig 8.28a

When an F factor (a plasmid) is transferred from a donor (F+) to a recipient (F–), the F– cell is converted to an F+ cell.

F+ cell F+ cell F+ cell F– cell

Replication and transfer of F factor

RECOMBINATION

Mating bridge Bacterial chromosome

F factor

The F (Fertility) Factor

§  Conjugation requires contact between donor (F+) cell and recipient (F-) cell

§  Genes for conjugation on F plasmid


Insert Fig 8.28b

When an F factor becomes integrated into the chromosome of an F+ cell, it makes the cell a high frequency of recombination (Hfr) cell.

F+ cell Hfr cell

Insertion of F factor into chromosome

Integrated F factor

Recombination between F factor and chromosome, occurring at a specific site on each

Hfr cells

§  Sometimes F factor integrates into chromosome §  F+ cell becomes Hfr (high frequency of

recombination)


Insert Fig 8.28c

When an Hfr donor passes a portion of its chromosome into an F– recipient, a recombinant F– cell results.

F– cell Hfr cell Hfr cell Recombinant F– cell

Replication and transfer of part of the chromosome

In the recipient, recombination between the Hfr chromosome fragment and the F– chromosome

Hfr Conugation

§  Conjugation between Hfr and F- transfers part of donor chromosome

§  Recipient is recombinant, but still F-

§  Why?


Plasmids

§  Conjugative plasmids: carry genes for plasmid transfer

§  Dissimilation plasmids: carry genes crucial for survival §  Catabolism of unusual compounds §  Toxins, bacteriocins (toxins that kill bacteria) §  Enhance pathogenicity

§  R factors: provide antibiotic resistance

Documents

Module 6 Microbial Genetics - University of Hawaii · 2014. 10. 20. · Genetics: science of heredity ! Study of what genes are, how they determine the characteristics of an organism,