Organisation And Control Of Prok Genome

Question 1

Features of prok?

Answer 1

• unicellular organism
• small in size 0.5-10Mb
• peptidoglycan cell wall
• contains 70s ribosomes
• does not contain membrane bound organelles
• no membrane system
• circular DNA not associated with histone proteins

Question 2

Prok genome consists of :

Answer 2

Bacterial chromosomes (found in nucleoid)
Plasmids (found in cytoplasm outside nucleoid)

Question 3

Features of Bacterial chromosome

Answer 3

• single circular DNA molecule associated with non-histone proteins
• found in nucleoid
• double stranded
• contains one origin of replication (Ori C )
• organised into operons under the control of a single promoter
• genes not interrupted by introns
• highly folded

Question 4

How is prok genome (bacterial chromosome) folded

Answer 4

Circular chromosome is folded into many loops - loop domains - bound to a central protein scaffold attached to the cell membrane
Each loop domain supercoil independently and is complexed with several DNA-binding proteins (specific supercoiling could affect the ability of the cell to express genes in that region)

Question 5

Features of plasmids

Answer 5

• smaller than bacterial chromosome
• double-stranded
• circular
• extra-chromosomal
• small plasmids may occur as multiple copies per cell (high copy number)
• has 1 origin of replication
• undergoes replication independently of chromosomal DNA
• genes present are not essential for survival and reproduction under normal conditions but are beneficial under stressful conditions
• eg. Genes for antibiotic resistance (R-factor), genes for conjugation (F-factor)

Question 6

How does bacteria reproduce?
Advantage?

Answer 6

Asexually via binary fission - 1 parent producing 2 identical daughter cells

Allows the bacteria to increase in number quickly to exploit the environment

Question 7

Outline the mechanism of binary fission

Answer 7

1. DNA attach to cell membrane
   
   • attaches at mesosome : a folded invagination of the membrane
2. DNA replication
   
   • starts at ori C, ends at termination sequence opp of ori C
   • occurs bidirectionally
   • supercoiling is removed with the help of enzyme gyrase
   • plasmids are replicated at the same time
3. Cell growth and division
   
   • cell elongates, the two circular DNA strands separate (still attached to cell membrane)
   • cell membrane folds inwards between the 2 DNA to form a double layer and new cell walls are secreted

(Note: distribution of plasmids may not be equal)

Question 8

Why do some offspring differ slightly from each other in their genetic make-up? (Prok)

Answer 8

due to spontaneous mutation in offsprings DNA (vertical transmission)
- eg. Insertions, deletions, base-pair substitutions

Horizontal transmission - transformation, transduction, conjugation

Question 9

What is vertical transmission?

Answer 9

Transfer of genes from mother to daughter cells
Mutation that occurs in parental DNA is inherited by daughter cells as the parent cell divides - gives rise to variation

Question 10

What is horizontal transmission?
What is genetic recombination?

Answer 10

Transfer of genetic material (DNA) from one bacterium to another that is not its offspring

Results in genetic recombination : combination of DNA from two individuals into the genome of a single individual

Question 11

What are the three types of horizontal transmission (+ definition) ?

Answer 11

Transformation : uptake of a foreign, naked DNA molecule by the cell from its surrounding environment

Transduction : transfer of bacterial DNA from one cell to another by means of a bacteriophage
- generalised transduction : any gene from bacterial host cell can be transferred to recipient cell
- specialised transduction : only a few specific genes can be transferred to recipient cell

Conjugation : direct transfer of DNA between 2 bacterial cells that are in contact with each other

Question 12

What is a minimal media?

Answer 12

Media that contains minimum nutrients possible for bacterial growth, without the presence of amino acids

Question 13

(Transformation)
What are competent cells? And how to make them?

Answer 13

Bacterial cells that can take up foreign DNA

(Technique in genetic engineering eg. insulin)
Artificial transformation : Cells can be made competent by introducing calcium ions and heat/electric shock

Question 14

Process of transformation

Answer 14

Foreign DNA are taken up by bacteria cells then incorporated into recipient DNA by genetic recombination
- bacteria cell will express the phenotype coded by foreign DNA

Question 15

Process of generalised transduction

Answer 15

(Lytic cycle)
Host cell’s degraded DNA/plasmid is packaged into new phage instead of viral DNA
Resultant virus then injects bacterial DNA into another bacterium but lytic cycle does not initiate
DNA from the first bacterium then may replace the homologous regions (same code) of the second bacterium (process similar to crossing over)

Question 16

Process of specialised transduction

Answer 16

Only occurs with certain types of bacteriophages - eg. Lambda phage
(Lysogenic cycle)
Viral DNA incorporates into host cell DNA (bacterial chromosome) at a specific site
When viral DNA excises itself during lytic phase, some bacterial DNA next to the integrated viral DNA is excised along with it
When the bacteriophage goes on to infect other cells, the bacterial DNA will be passed to the cell along with viral DNA

Question 17

(Conjugation)
What defines whether a bacterium is a donor or a recipient?

Answer 17

Donors (male / F+ strain) : have F plasmid which codes for the production of sex pili (plural) (singular : pilus)
Recipients (female / F- strain) : without F plasmid

Question 18

Process of conjugation

Answer 18

1. Donor cell attaches to recipient cell with its sex pilus which acts like a grappling hook drawing the cells together
2. The cells come into contact and a cytoplasmic bridge is formed to allow DNA transfer
3. In donor cell, F plasmid is nicked at origin of transfer and 1 DNA strand (tDNA) is separated and exported to the recipient cell via the cytoplasmic bridge
4. The single stranded tDNA strand acts as a template for synthesis for its complementary strand to form the double stranded plasmid - the recipient now contains an F plasmid and becomes a donor
   The remaining strand in the donor cell also synthesises its own complementary strand

Question 19

What are R plasmids? (Can also result in conjugation)

Answer 19

Plasmids that carry genes that code for enzymes that confers resistance to antibiotics

Have genes coding for production of sex pili, like F plasmid and are therefore capable of transferring their genes to other bacteria cells by conjugation

Question 20

What is gene expression and regulation?

Answer 20

Gene expression : transcription and translation of a gene into a functional product
Gene regulation : the control of whether a gene is expressed and the level of its expression (high or low)
- allows certain genes to be turned on when their products are required and off when not needed to save energy and resources

Question 21

(Overview)
How does prok regulate their gene expression ?

Answer 21

• (mainly) regulation at transcriptional level : genes that code for proteins/enz that are involved in a single pathway are grouped together under the control of a single promoter allowing all of the structural genes to be controlled simultaneously
• regulation using environmental signals to help them respond (quickly) to changes in the environment
  eg. Presence of a substrate (lactose) - lac operon
  Presence of an end product (tryptophan) - trp operon

Question 22

What are the components of an operon?

Answer 22

(Re-POST)
(Re)gulatory gene : codes for repressor protein
- active = prevent transcription by blocking RNA polymerase from accessing and transcribing the genes, operator is switched off
- inactive = cannot bind to operator, RNA polymerase able to transcribe genes, operator is switched on

(P)romoter : where RNA polymerase binds to initiate transcription

(O)perator : acts like a molecular switch allowing the turning off and on of genes, controls the access of RNA polymerase to the genes

(S)tructural genes : codes for specific proteins with related functions in the same pathway
- transcribed to form a single polycistronic mRNA : mRNA containing genetic info for synthesis of more than one polypeptide, punctuated with stop and start codons for individual polypeptides

(T)erminator

Question 23

What are the two types of operons?

Answer 23

Inducible operons : usually turned off but can be induced to turn on in response to a specific molecule resulting in synthesis
- eg. lac operon induced in the presence of lactose (specifically allolactose)

Repressible operons : usually turned on but can be turned off in response to a specific molecule, stopping synthesis
- eg. trp operon is repressed in the presence of tryptophan

Question 24

What is negative and positive control of operons?

Answer 24

Type of control : defined by response of the operon to specific regulatory proteins (repressor/activator)

Negative control - genes in the operon are switched off by active form of repressor protein
Positive control - genes are expressed only when an active form of an activator is present
(An operon can be under both types of control eg. lac operon)

Question 25

What is a lac operon?

Answer 25

A cluster of structural genes coding for enz involved in metabolism of lactose (Catabolic)

Question 26

What are the components of a lac operon?

Answer 26

(Re)gulatory gene : lac i gene - codes for active lac repressor molecule (quaternary protein with two identical polypeptides) with two recognition sites - repressor molecule bound to operator prevents transcription (negative control) CAP binding site : where cAMP-CAP complex binds (positive control) (P)romoter (O)perator (S)tructural genes : lacZ - code for B-galactose (enz thatbreaks down lactose into glucose and galactose) lacY - code for lactose permease (enz that tpt lactose into bacterial cell) lacA - code for galactoside transacetylase (enz needed in lactose metabolism)

Question 27

What are the two recognition sites of the lac repressor molecule

Answer 27

DNA-binding site : recognises specific DNA sequence of lac operon operator Allosteric site : binds with allolactose inducer and similar molecules

Question 28

What happens in the ABSENCE OF LACTOSE (in env) ? Why is there still small amounts of enz being synthesised? (Negative control)

Answer 28

No lactose = no allolactose Active repressor molecule binds to operator and blocks RNA polymerase, preventing transcription (negative control) Lac operon is leaky - active repressor does not bind permanently to operon and can detach, allowing binding of RNA polymerase to promoter to initiate transcription

Question 29

What happens in the PRESENCE OF LACTOSE (in env) ? Why is lac operon an inducible operon ?

Answer 29

Lactose enters cells through lactose permease, coded for by lacY (produced because lac operon has a minimal level of transcription) Allolactose (an isomer of lactose) acts as an inducer by binding to the allosteric site of the repressor molecule - changes 3D conformation of repressor and prevents it from binding to operator RNA polymerase can then bind to promoter and start transcribing - genes are switched on Inducible operon as Enz coded for by lac genes are only produced in response to particular substrate (allolactose inducer) - produced only when needed

Question 30

Why is glucose important in expression of lac operon ? And why does glucose concentration (in env) affect expression of lac operon?

Answer 30

RNA polymerase does not bind efficiently to the promoter and hence synthesis of the 3 enz are low even in presence of high lactose concentration therefore need activator protein which increases rate of transcription - catabolite activator protein (CAP) Glucose is the preferred substrate for bacteria - it can be used directly for respiration (monosaccharide) while lactose has to be hydrolysed

Question 31

What happens in the presence of HIGH GLUCOSE CONCENTRATION (in env) ?

Answer 31

Transport of glucose into the bacterial cell inhibits the enzyme adenyl cyclase which converts ATP into cyclic AMP (cAMP) cAMP level is low, catabolite activator protein (CAP) is inactive and cannot bind to CAP binding site, RNA polymerase cannot bind to promoter efficiently If lactose is present : lac operon is switched on but expression is basal (low)

Question 32

What happens in the presence of LOW GLUCOSE CONCENTRATION (in env) ? (Positive control)

Answer 32

Low glucose concentration, less adenyl cyclase is inhibited, more ATP is converted into cAMP cAMP binds to CAP to form cAMP-CAP complex which binds to CAP binding site (positive control) This enhances the binding of RNA polymerase to lac promoter and increases the expression of genes If both glucose and lactose present : operon will not be maximally expressed until all the glucose is metabolised

Question 33

When does maximal expression of the lac operon occur?

Answer 33

In the absence of glucose and presence of lactose

Question 34

What control is lac operon under? What type of operon is the lac operon?

Answer 34

Dual control - negative control by lac repressor - positive control by cAMP-CAP (activator) Inducible operon - turned on by the presence of allolactose

Question 35

What is a trp operon?

Answer 35

Trp operon regulates the biosynthesis of tryptophan, an important amino acid (Anabolic)

Question 36

What are the two levels of control tryptophan (end product) biosynthesis pathway shows ?

Answer 36

1. If tryptophan level is high, it can inhibit the first enz in the biosynthesis pathway (regulation of enz activity) 2. If the abundance of tryptophan continues, the cell can stop synthesising more enz in the pathway by blocking transcription of the genes for the enz (regulation of gene expression)

Question 37

What are the components of trp operon?

Answer 37

(Re)gulatory gene : trpR - codes for an inactive repressor protein (P)romoter (O)perator (S)tructural genes : trp E, trp D, trp C, trp B, trp A - all encodes enz that catalyse tryptophan synthesis (T)erminator

Question 38

What happens in the presence of LOW TRYPTOPHAN CONCENTRATION (in growth medium) ?

Answer 38

Low tryptophan concentration = repressor protein is inactive and is not bound to operator region RNA polymerase can bind to promoter and initiate transcription

Question 39

What happens in the presence of HIGH TRYPTOPHAN CONCENTRATION (in growth medium) ?

Answer 39

Tryptophan acts as a co-repressor to turn off transcription - it binds to and activates the repressor so that the repressor-tryptophan complex can bind to the operator and block transcription (negative control) RNA polymerase cannot bind to promoter and biosynthesis of tryptophan is stopped since enz are not produced

Question 40

What is the type and control of the trp operon?

Answer 40

Repressible operon - synthesis of enz can be turned off by presence of end product (tryptophan) Negative control - genes are not transcribed when the repressor is bound

Question 41

What are the advantages of operons in bacteria?

Answer 41

Allows for the regulation of gene expression so bacteria only synthesis enz required - no wastage of energy and resources Allow bacteria to use a variety of sugars or substrates present in the environment - respond to changes in the environment

Question 42

What are the two types of metabolism?

Answer 42

(ABCD) Anabolic Build Catabolic Destroy

Question 43

What are the criteria to differentiate transformation, transduction and conjugation?

Answer 43

the DNA being transferred - naked pieces of DNA, random pieces, plasmid involvement of an agent - virus special criteria - both bacteria must be in contact