Chapter 8: Microbial Genetics - Part I: Gene Regulation & Mutations

Where do bacterial regulate gene expression

during transcription

In Prokaryotes Operons are:

one promoter and terminator regulates gene expression for a cluster of gene known as operons

Constitutive Genes in Bacteria:

• 60-80% of genes are constitutive : products are continuously produced

  • genes are “ON” all the time

  • codes for proteins/enzymes that are needed in large amount for essential processes like in cellular respiration and binary fission

Repression

regulatory mechanism that inhibits gene expression, and decreases the synthesis of a protein

• repressor proteins

• turning OFF

Induction

regulatory processes that turns on (induces) the transcription of a gene(s), increasing protein synthesis

• inducers are substances that initiate transcription

• turning ON

Promoter

initiation of transcription → RNA polymerase binding

Operator

regulatory element that controls the activation or repression of the operon

• transcription factors can bind this region or to adj. binding sites to activate or repress transcription

Operon

promoter and group (cluster) of genes that code for enzymes and proteins in bacteria - regulated as a unit

Inducible Operon

not usually transcribed, must be induced and activated

• default is OFF, you have to induce operon → turn ON

Repressible Operon

continuously transcribed until repressed

• default is ON, needs to be repressed (turned OFF)

Transcription factors

regulatory proteins that bind DNA and affect transcription

1. Repressors

2. Activators

Repressors & How they work

• proteins known as transcription factors that can bind to DNA during transcription

• block the ability of RNA Pol to bind

• OFF

• do not need inducer to repress

Activators & How they work

• proteins known as transcription factors that can bind to DNA during transcription

• promote transcription of genes by facilitating binding of RNA Pol

  • think if it as a signal that recruits RNA Pol

• ON

• DO NOT WORK if inducers are not present

Genetic Inducers

Induce transcription by:

• removing/blocking repressors

• allowing the binding of activators

• work by binding to transcription factors NOT operon

• induce transcription ALWAYS

Repressor + Inducer =

ON Operon:

WHY:

• removal of repressor so there is no blockage

Activator + Inducer =

ON Operon

WHY:

• recruitment of RNA Pol to promoter

Lac Operon

contains genes needed for the transport and catabolism of lactose

• 3 genes coordinately regulated as a unit

• genes code to allow bacteria to use lactose

Lac Operon is an Inducible Operon meaning it is expressed when:

1. Glucose is absent or in low concentrations

2. Lactose is present

• usually OFF , meaning it is NOT essential

• as long as there is glucose Lac operon is off that is why it is inducible

Lac Operon Transcription Factors

1. Activator - CAP

2. Repressor - Lac repressor = blockage

Lac Operon Inducers

Bind to transcription factors

1. CAMP → binds to activator

2. Allolactose/lactose → binds repressor

Lactose is absent , is Lac Operon ON or OFF ?

OFF

• repressor is active, so operon is off

• the repressor protein binds with operator, preventing transcription from the operon

Lactose is present , is Lac Operon ON or OFF ?

ON

• repressor is inactive, operon is on

• the inducer allolactose binds to the repressor protein, inactivating the repressor and it can no longer block transcription

What controls cAMP ?

Glucose controls the intracellular levels of cAMP (alarm signal)

• cAMP signals glucose is low

Glucose is present No Lactose Is the Operon OFF or ON ?

OFF

• Operon is off because glucose is present , so low cAMP (inducer) levels meaning CAP (activator) can not bind

• repressor is bound to operator blocking polymerase

Glucose Present Lactose Present Is the Operon OFF or ON ?

OFF

• THINK: Glucose is present so we don’t need Lac Operon

• Because glucose is present, cAMP is low and CAP cannot bind

• The Lactose/Allolactose function as inducer and are present so repressor is NOT repressing but there is glucose & no inducer cAMP for activator CAP so it is OFF

No Glucose Lactose Present Is the Operon OFF or ON ?

ON

• Glucose is not present which means cAMP is high, cAMP is the inducer for the activator CAP meaning cAMP binds to CAP & allows it to bind to the activator-binding site

• Allolactose & lactose is present as well which is the inducer for the Lac operon repressor, meaning it binds to the repressor which prevents it from binding to the operator and blocking polymerase

No Glucose & No Lactose Is the Operon OFF or ON ?

OFF

• Think: Lac Operon functions to make genes needed for the transport and catabolism of lactose , if there is no lactose why would it be on ?

• There is no glucose so cAMP meaning it can bind to CAP & then to the operon to recruit RNA Polymerase however there is no lactose/allolactose to induce the repressor thus the repressor is bound to the operon blocking polymerase

Down-Regulation: Lac Operon OFF

1. Glucose present, Lactose absent

2. Glucose present, Lactose present

3. Glucose absent, Lactose absent

Up-Regulation: Lac Operon ON

1. Glucose absent, Lactose present

What does this graph represent ?

This represents growth of cells on glucose OR lactose alone

• notice they grow on glucose much faster than on lactose

What does this graph represent ?

Growth on glucose & lactose combined

• Glucose is used first, then when it is all consumed there is a lag which represents the operon turning on, then lactose is used

What is a mutation

any change in the base sequence of genes

What is a point mutation ?

a single nucleotide base change

• base substitution: single nucleotide base is replaced

What are mutagens

agents that can cause mutations

What does wild type refer to ?

without mutation; the normal state of an organism (also used for genes)

What is a missense mutation ?

change that codes for a different amino acid

What is a nonsense mutation ?

changes amino acid codon into STOP codon causing truncation (cut short)

What is a silent mutation ?

Does not change amino acid

• there is a change in DNA → codon but redundancy allows it to code for same amino acid

What is a frameshift mutation ?

Changes reading frame of codons, shifting it and creating new codons

What type of mutation is depicted ?

Missense

What type of mutation is depicted ?

Nonsense

What type of mutation is depicted ?

Silent

What type of mutation is depicted ?

Frameshift

Causes of Mutations ?

1. Spontaneous mutations

2. Induced mutations

What are spontaneous mutations ?

introduced by errors in replication and/or repair, and by recombination events

• proof-reading mechanisms for prevention

• rare events that could be beneficial allows organisms to survive and adapt to env.

• Recombination: long stretches of DNA move in chromosomes, introducing frameshifts

What are induced mutations ?

Result from exposing DNA to physical or chemical agents (mutagens)

• NOT natural

Nucleotide altering chemicals

alter structure of nucleotides, altering base pairing

Nucleotide analogs

compounds structurally similar to normal nucleotides; act like nucleotides in DNA synthesis

Intercalating agents

insert between base pairs resulting in frameshift mutation

Physical Mutagens

1. Ionizing radiation

2. Non-ionizing radiation

Ionizing Radiation & Effects

• x-rays and gamma rays

• can cause breaks in the backbone of DNA, separates strands by breaking H bonds, damages base structure

• irreversible damage

Non-ionizing Radiation & Effects

• UV radiation

• causes thymine (pyrimidine) dimers

What are Thymine Dimers ?

Adj. thymines become cross-linked, forming a thymine dimer and disrupt their normal base pairing

• double covalent bond

What are the two ways pyrimidine dimer’s can be repaired ?

1. Photoreactivation → photolysases

• ONLY BACTERIA

2. Nucleotide excision repair (dark repair) → enzyme complex

Photoreactivation / Light Repair

An enzyme requiring visible light for activity breaks covalent bond joining adj. thymine molecules allowing them to resume original shape

Excision Repair

1. Endonucleases and exonucleases cut out the damaged DNA

2. resynthesis of original DNA by DNA Polymerase I

3. Ligation to repair sugar phosphate backbone by DNA ligase

Horizontal Gene Transfer

lateral transfer to cells of the same generation

1. Transformation (recombination)

2. Transduction (recombination)

3. Conjugation (with or w/o recombination)

Genetic Recombination

Exchange of genes between 2 DNA molecules to form new combinations of genes

Griffith’s Classic Experiment

Demonstrated bacterial transformation, where heat-killed lethal bacteria transformed living non-lethal bacteria into lethal ones. This proved that genetic information could be transferred between organisms, leading to the discovery of the "transforming principle"

Transformation

*EXOGENOUS DNA

1. Recipient cell takes up donor DNA

2. Donor DNA aligns with complementary bases

3. Recombination occurs between donor DNA and recipient DNA

Competence

alterations in the cell wall that make it permeable to DNA

• a cell is competent when it is able to acquire exogenous DNA

Transduction

Transfer of DNA between different bacteria by a virus

1. a phage infects the donor bacteria cell

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

3. Occasionally during phage assembly pieces of bacterial DNA are paackaged in phage capsid → phage then lyses cell

4. Phage carrying bacteria DNA infects new host cell , the recipient cell

5. Recombination can occur producing a recombinant cell with a genotype diff. from both donor and recipient cell

Conjugation

transfer of DNA between two bacteria

• can transfer plasmid OR portion of the chromosome

Transformation VS. Transduction VS. Conjugation Simply Put

Transformation

• exogenous DNA

Transduction

• Virus

Conjugation

• sharing plasmid or portion of chromosome
  

Plasmids

• self-replication, circular DNA molecules

• extra chromosomal DNA

• can aid bacterial survival

• R factors: plasmids that code for antibiotic resistance genes

F factor

a plasmid

F + cell

donor cell

F - Cell

recipient cell , converted to F+cell

Genetic recombination

An Hfr cell (High-frequency recombination cell) is a bacterium with a fertility factor ( factor) plasmid integrated into its main chromosome. Unlike normal cells, Hfr cells attempt to transfer their entire chromosome to recipient cells during conjugation, often transferring bacterial genes instead of just the plasmid, creating high genetic diversity.