bacteria

Question 1

Outline the process of Binary Fission

• ori
• unzipping, ; formation of replicaiton bubble
• template
• complementary base pairing
• move to opp poles
• attach to plasma membrane
• elongates
• circular with no free end, interlock
• topoisomerase
  invagination

Answer 1

1.DNA replication begins at the origin of replication** (ori) where DNA is unzipped by breaking hydrogen bonds between bases of the 2 strands to form a replication bubble
2.DNA replicates by semi-conservative replication** where each original strand serves as template** for synthesis of daughter strands by complementary base pairing*
3. 2 newly formed ori move to opposite poles of the cell and attach to the plasma membrane
4.Cell elongates to prepare for division.
5.DNA is circular with no free ends, and the 2 daughter DNA molecules will be interlocked with the completion of replication.
6.Enzyme topoisomerase cut, separate and reseal the two DNA molecules
7.Invagination** of the plasma membrane and the deposition of new cell wall (division septum) eventually divide the parent cell into two daughter cells
➔each inherits a complete genome (genetically identical)

Question 2

Outline the process of bacterial conjugation

Refers to the direct transfer* of genetic material from one bacterial cell to another **, by cell-to-cell contact through a temporary link* between the 2 cells

• transfer of DNA is always one way —> from donor cell to recipient cell
  On the F plasmid, there is a segment of DNA called an F factor that carries genes coding for sex pili
  Due to the presence of F factor, the donor cell is able to produce appendages called sex pili to attach itself to the recipient cell

Answer 2

1. Sex pilus** (coded for by F factor)of F+ bacterial cell makes contact* with a F-cell and retracts* to bring the 2 cells closer
2. The hollow pilus then acts as a cytoplasmic mating bridge** between the 2 cells
3. One of the 2 strands of the plasmid DNA is nicked and transferred from the F+ cell to the F-cell* through the bridge while the other strand used for template for elongation*
4. The single stranded F plasmid DNA circularises in F-cell and is used as a template** by DNA POLYMERASE** to synthesise a complementary strand for a double-stranded* plasmid DNA. The F-recipient cell is now a F+cell

5.Replication of the plasmid occurs via rolling circle DNA replication

Question 3

Describe the Rolling Circle DNA replication

Answer 3

a)ONE STRAND of double stranded F plasmid is nicked by a nuclease
➔free 3’OH end is then used as a primer for strand elongation by DNA polymerase** using the UNNICKED/intact strand as a template**
➔elongation process is facilitated by the displacement of the 5’ end of the nicked strand and is transferred across the cytoplasmic mating bridge to the recipient bacterium
➔Upon completion of a unit length of the plasmid DNA (after 1 round), another nick occurs to release the original strand

b)In the recipient cell, the single strand of F plasmid DNA re-circularises and serves as a template for the synthesis of a complementary daughter strand to form a double stranded circular DNA.

Question 4

Outline the process of bacterial transformation [2m]

Answer 4

1. Fragments* of foreign naked DNA* (from lysed bacterial cells) in the surrounding medium are taken up by a bacterial cell via surface proteins*
2. The foreign DNA is incorporated into the bacterial chromosome/DNA* via CROSSING OVER/HOMOLOGOUS RECOMBINATION**
3. If the foreign DNA contains a different allele that is now expressed in the bacterial cell, the bacterial cell has transformed* –> permanent change in genotype and phenotype

(extra)
- recombinant DNA will be passed on to all subsequent offspring through binary fission

Question 5

Suggest 2 potential benefits of conjugation for the recipient bacteria

Answer 5

1. Gains new alleles that, when expressed, allow it to survive different environment, i.e Antibiotic resistance
2. use of a new metabolite/resources (e..g new carbon source by producing the relevant enzyme)

Question 6

Outline the stages in general bacterial transduction

** means the WORDS MUST APPEAR

Answer 6

1. A phage* infects a bacterium, injecting its viral genome (DNA) into the host cell
2. The bacterial DNA is degraded into small fragments, one of which may be randomly packaged* into a capsid head* during the spontaneous assembly* of new viruses
3. Upon cell lysis, the defective phage will infect another bacterium and inject bacterial DNA from the previous host cell into the new bacterium
4. Foreign bacterial DNA can replace the homologous region* in the recipient cell’s chromosome through homologous recombination***, allowing the expression of a different allele from the previous host

Question 7

Outline the stages in specialised bacterial transduction

Answer 7

1. A TEMPERATE PHAGE* infects a bacterium, injecting its viral genome into the host cell
2. The viral DNA is integrated into bacterial chromosome* forming a prophage***
3. which may be improperly excised* to include adjacent segment* of bacterial DNA* and not the entire phage DNA DURING AN INDUCTION** event
4. Hence phage-bacterium hybrid DNA may be packaged into a capsid head* during the spontaneous assembly*** of new viruses
5. Upon cell lysis, the defective phage will infect another bacterium and inject bacterial DNA from the previous host cell into the new bacterium
6. New alleles from the previous bacterial cell can be incorporated into the genome of the new host* by homologous recombination** or integration of phage-bacterium hybrid DNA as defective phage enters the lysogenic cycle, allowing the expression of a different allele from the previous host

Question 8

qn: A mutant bacterium was found to contain a defective Lac I gene. When such mutant bacteria were placed in lactose containing medium, lactose levels did not change.
Suggest an explanation for this scenario [2m]

Recall: lac i gene:codes for lac repressor
Lac repressor hasDNA binding site(binds to operator)and allosteric site (binds to allolactose)

Answer 8

1. allolactose unable to bind to defective repressor protein thus there is no conformational change of repressor as it continues to bind to the operator
   OR
   1.. Repressor** binds to irreversibly to operator
2. hence lac operon is permanently switched off and thus beta-galactosidase** not produced hence lactose not broken down

Question 9

Explain the role of the F plasmid [3m]

Answer 9

1. F factor on F plasmid codes for proteins* necessary for the formation of sex pili* and subsequent cytoplasmic mating bridge*
2. allowing for conjugation* to occur between bacteria
3. this allows for bacterial genes* to be transferred between bacteria* and hence increase genetic variation* between bacteria

Question 10

qn: the regulatory gene of the tryptophan is located far away from the trp operon but it can still regulate expression of the trp operon. Why?

Answer 10

1. R codes for the trp repressor** which carries out the repression of the operon
2. trp repressor is DIFFUSIBLE PRODUCT which can diffuse to the location of the operator*** to bind to it and exert its effect

Question 11

qn: explain how the high levels of tryptophan affects expression of the trp operon. [3m]

Answer 11

1. tryptophan binds to the allosteric site of the trp repressor, to change the repressor to its active form
2. active repressor binds to the operator** which prevents the binding of RNA POL** to promoter**
3. hence switching off the operon/ preventing expression of operon

Question 12

explain what is meant by the term: structural gene

Answer 12

structural gene is any gene that codes for a protein product that has an enzymatic function in a metabolic pathway *

Question 13

explain what is meant by the term: regulatory gene

Answer 13

regulatory gene codes for a protein* involved in regulating expression of structural genes*

Question 14

Using the (context:) ara operon, explain what is meant by the term ‘operon’

Answer 14

1. Operon is a cluster of genes, ara a, ara b and ara c,under the control of the same promoter* and operator
2. allow for functionally related proteins to be synthesised as a unit, arabinose** can be broken down

Question 15

Explain what is meant by a polycistronic mRNA

Answer 15

A messenger mRNA that contains the base sequence coding for the amino acids sequence of several proteins

▪A single mRNA contains multiple start codons (AUG) and stop codons (UAG, UAA, UGA) (one per polypeptide e.g. 3 sets for lac operon, 5 sets for trp operon)

▪Gives rise to a total of ___ different polypeptides which can be translated from a single mRNA, illustrating the polycistronic nature of the mRNA

Question 16

What is a regulator gene?

Answer 16

Any of several kinds of nucleotide sequences involved in the control of the expression of structural genes
▪Codes for a protein involved in regulating the expression of other genes e.g. repressor, CAP

▪Has its own promoter and terminator sequences
▪Not within operon, usually far away, but gene products that control the expression are diffusible*

Question 17

What is a structural gene?

Answer 17

Any gene that codes for a protein product that forms part of a structure or has an enzymatic function

Question 18

What is an effector?

Answer 18

a small molecule that binds to a specific protein, causing a conformational change and hence regulating its biological activity
E.g. Inducer - allolactose in lac operon
E.g. Corepressor - trptophan in trp operon

Question 19

Advantages of regulation

Answer 19

1. Operon contain group of genes under the control of the same promoter that allow for functionally related proteins to be synthesised as a unit;
2. An operon can be turned ‘on’ or ‘off’ according to certain changes/conditions so as to respond rapidly and appropriately to the environment
3. Bacterium only produces enzymes when required allowing the bacteria to make economical use of energy and resources/conserve resources i.e. relevant genes are expressed only when necessary;
4. Especially since bacteria are able to use a variety of metabolites e.g. glucose is metabolised preferentially over lactose, thus not economical to produce lac genes in the presence of glucose;
5. The abovementioned provide a selective advantage to such bacteria

Question 20

Structure of Lac Operon

Answer 20

A cluster of 3 structural genes
▪lacZ codes for beta-galactosidase: enzyme that hydrolyses lactose into glucose and galactose

▪lacY codes for permease: facilitates movement of lactose from outside of cell to inside of cell

▪lacA codes for transacetylase: function remains unknown

▪Promoter➔RNA polymerase binding site

▪Operator ➔lac repressor binding site

• Operator overlaps with promoter
• Catabolite Activator Protein (CAP) binding site within promoter

Question 21

Regulatory gene of lac operon

Answer 21

Lac I gene - that codes for lac repressor*

Lac repressor has DNA binding site (binds to operator) and allosteric site (binds to allolactose)

Question 22

What type of operon is lac operon?

What type of operon is trp operon?

Answer 22

Inducible Operon

Repressible operon

Question 23

What is the purpose of the lac operon

Answer 23

regulates the production of inducible enzymes** such as Beta-galactosidase and other proteins involved in the breakdown of lactose**

Question 24

What type of regulation does lac and trp operon have?

Answer 24

(lac) Dual regulation - negative regulation by the lac repressor and positive regulation by CAP
(trp) negative regulation by the trp respressor

Question 25

What is the effector of lac operon and trp operon? And what are their effects on the operon?

Answer 25

(lac) Inducer lactose --> turns on transcription of structural genes VS (trp) corepressor tryptophan --> turns off transcription of structural genes

Question 26

What are the types of metabolic pathway that the lac and trp operon takes?

Answer 26

(lac) Catabolic pathway; breakdown metabolites | (trp) Anabolic pathway : synthesise metabolites

Question 27

Default state of the lac operon and the trp operon

Answer 27

(lac) off | (trp) on

Question 28

Describe the regulation of lac operon. - in the absence of lactose - in the absence of glucose

Answer 28

1. In the absence of lactose, a basal level of beta galactosidase and permease is present in the cell: repression of lac operon by lac repressor is leaky ( as the binding of the repressor is mediated by weak interactions) 2. Lactose enters** the cell by permease** 3. And is converted to allolactose** by beta-galactosidase** 4. Allolactose acts as an inducer** and binds to allosteric site of lac repressor**. This causes a change in conformation** lac repressor and lac repressor becomes inactive** 5. The inactive repressor and can no longer bind to operator of lac operon** (no longer complementary in shape and charge) 6. Promoter ** site available for RNA polymerase** to bind 7. When glucose is absent ➔high levels of cAMP is present ➔cAMP binds to allosteric site of CAP and activates CAP ➔activated CAP binds to promoter of lac operon which increases the affinity** of RNA polymerase** to the promoter** 8. Transcription frequency of structural genes lacZ, lacY and lacA to produce beta-galactosidase, permease and transacetylase respectively to breakdown lactose thus increases. 9.(Give one example of a product and what it does) Note: there will be a time lag for lac operon genes to be expressed –time taken for transcription of genes and subsequent translation to form gene products

Question 29

What conditions allow lac operon to be ON

Answer 29

- presence of lactose | - absence of glucose

Question 30

Describe the structure of Trp Operon

Answer 30

A cluster of 5 structural genes: trpE, trpD, trpC, trpBand trp A ▪Code for enzymes in tryptophan biosynthesis pathway ▪5 genes ➔5 polypeptides ➔3 enzymes (2 of the enzymes are dimers, trpA+trpB & trpD+trpE & trpC on its own) ▪Promoter ➔RNA polymerase binding site ▪Operator ➔binding site for trp repressor complex with trp Operator within promoter

Question 31

What is the regulatory gene of trp repressor?

Answer 31

Trp R gene that codes for trp repressor* | Trp repressor has DNA binding site to bind to the operator and allosteric site to bind to the tryptophan

Question 32

What is the purpose of the trp operon?

Answer 32

it regulates the production of repressible enzymes** for the synthesis** of the amino acid tryptophan **

Question 33

Describe the regulation of trp operon

Answer 33

1. When tryptophan present in high concentrations in the cell 2. Tryptophan acts as a corepressor** and binds to the allosteric site of trp repressor* 3. this causes a change in the conformation** of trp repressor and trp repressor becomes active** 4. the active repressor* can bind to the operator** 5. prevents the binding of RNA pol** to promoter** 6. prevents transcription of structural genes** and expression of operon 7. synthesis of tryptophan** is stopped

Question 34

What happens when there is a mutation in the lacZ structural gene? observation: truncated/ non-functional beta-galactosidase

Answer 34

permease and transacetylase coded by lacY and lacA will still be produced as the enzymes are translated independently because each has its own start and stop codon on the polycistronic mRNA

Question 35

" Enzymes not present even when lactose is present " --> indicates that the operon is switched off What has happened?

Answer 35

Possible location of mutations : promoter or lac I regulatory gene lac I: - change in conformation of DNA binding site of repressor --> binds irreversibly to/ cannot dissociate from operator - change in conformation of allosteric site of repressor --> cannot bind to allolactose (inducer) to become inactivated --> remains bound to the operator mutation in promoter sequence: - RNA pol cannot bind to promoter --> no transcription

Question 36

" enzymes always present even when lactose is absent" suggest: genes in operon are constitutively transcribed What has happened?

Answer 36

Mutation in the lac I or promoter sequence lac I: ▪Truncated/ non-functional/ absent repressor ▪Conformational change in DNA binding site of repressor ➔cannot bind to operator ▪(for trp operon) Conformational change in allosteric site of repressor ➔cannot bind to corepressor to form repressor complex ➔ cannot bind to operator operator: Repressor unable to bind to operator ➔RNA polymerase can continue to bind to promoter & transcribe structural genes

Question 37

Describe 2 ways in which the structure of polycistronic mRNA molecule produced when the lac operon is expressed differs from the pre-mRNA transcripts found in eukaryotes

Answer 37

1. Polycistronic mRNA has several start and stop codons while eukaryotic premrna transcripts only have 1 start and 1 stop codon 2. Eukaryotes pre-mRNA transcripts intron sequences while polycistronic mRNA do not have introns

Question 38

Explain how operons allow for rapid response by bacteria to environmental change. [2m]

Answer 38

1. multiple genes* coding for products involved in the same biochemical pathway are under the control of a single promoter** 2. these genes are therefore turned "on" or turned "off" together, allowing the bacteria to respond rapidly to changes in the environment

Question 39

Describe the role of the tra operon polycistronic mRNA [2]

Answer 39

1. it is the template for the translation** of functionally related proteins involved in conjugation 2. Such that the 34 structural genes**(in context of the qn) are synthesised together **from a single mRNA 3. this allows the E coli to make economical use of energy and resources**