Organisation and Control of Prokaryotic Genome

Question 1

(d) Describe the structure and organisation of prokaryotic genome (including DNA/RNA, single-/double-stranded, number of nucleotides, packing of DNA, linearity/circularity and presence/absence of introns)

Answer 1

Most bacteria possess a protective cell wall, which contain peptidoglycan.
(i) Gram-positive bacteria
- Have cell walls with a relatively large amount of
peptidoglycan
(ii) Gram-negative bacteria
- Have less peptidoglycan in their cell walls
- Have an outer membrane that contains
lipopolysaccaharide.

Most bacterial species contain a large, circular chromosome.
- The chromosome inside a bacterial cell is highly
compacted and found within the nucleoid and is not
bounded by a membrane
- A typical chromosome is a double-stranded DNA (a
few million base pairs in length) that is associated with
DNA binding proteins (non-histone scaffolding
proteins).
- Most bacterial species contain a single type of
chromosome, but it may be present in multiple
copies.
- Several thousand different genes are interspersed
throughout the chromosome. Structural genes (i.e.
sequences that encode proteins) account for majority
of bacterial genome. [NO Introns cus too small, only
non-coding sequences are: Promoter, other
regulatory gene sequences such as operator and
terminator)
- The non-coding sequences are located between
adjacent genes, i.e. intergenic sequences.
- Each chromosome possesses only one origin of
replication. The origin of replication is a sequence
that functions as an initiation site for the assembly of
several proteins that are required for DNA
replication.
- In addition to the chromosome, bacteria also have
several plasmids, which are small, circular pieces of
DNA that exist independently of the bacterial
chromosome
–> Plasmids are self-replicating, i.e. their
replication is independent of the bacterial
chromosome, as it contains its own origin of
replication.
–> Plasmids are not necessary for survival of
bacteria. However, in many cases, certain
genes within a plasmid confer advantages to
bacteria’s survival in stressful environments
1. Fertility plasmids (F plasmids), also known as F
factors, allow bacteria to mate with each other.
Through the mating process, F plasmid
facilitates genetic recombination, which may
be advantageous in a changing environment
that no longer favours existing strains in a
bacterial population
2. Resistance plasmids, also known as R factors,
contain genes that confer resistance against
antibiotics and other types of toxins.

Structure of Bacterial Chromosome:
To fit within the bacterial cell, the chromosomal DNA must be compacted several folds. Part of this compaction process involves the formation of loop domains. A loop domain is a segment of chromosomal DNA that is folded into a structure that resembles a loop. DNA binding proteins anchor the base of these loops.
- Supercoiling leads to further compaction of the looped bacterial chromosome.
–> The chromosome in living bacteria is negatively
supercoiled. The force of negative supercoiling
may promote DNA strand separation in small
regions, enhancing genetic activities such as
replication and transcription.
–> DNA gyrase and topoisomerase I control the
degree of supercoiling in bacterial chromosome.

Question 2

(g) Outline the mechanism of asexual reproduction by

binary fission in a typical prokaryote

Answer 2

• Before the bacterial cell divides, semi-conservative
  replication of parental DNA begins at the origin of
  replication to give rise to two origins.
• As the chromosome continues to replicate, each
  origin moves rapidly toward the opposite end of
  the cell and adhere to the cell surface membrane.
• While the chromosome is replicating, the cell
  elongates. Elongation of the cell also separates the
  two identical copies of the chromosomes.
• When replication is complete and the cell has
  reached about twice its initial size, its cell surface
  membrane invaginates, and deposits new cell wall
  materials. Two daughter cells are formed which are
  genetically identical to the parent cell. Each cell
  inherits a parental strand of DNA.
• If the bacterial cell contains plasmids, these will
  replicate independently of the bacterial
  chromosome.
  When a plasmid is found in multiple copies in a cell,
  binary fission usually results in each daughter cell
  containing one or more copies of the plasmid.

Question 3

Describe how transformation give rise to variation in prokaryotic genomes

Answer 3

Transformation

Transformation is the alteration of a bacterial cell’s genotype by the uptake of naked, foreign DNA from the surrounding environment.
Usually, this DNA was released into the environment when another bacterium had died. Many bacteria possess cell-surface proteins (COMPETENCE factors) that recognize and transport DNA from closely related species into the cell. This foreign DNA can then be incorporated into the genome, either by integration or recombination via crossing over at homologous regions.
The cell is now a recombinant: its chromosome contains DNA derived from two different strains.

Question 4

Describe how transduction give rise to variation in prokaryotic genomes

Answer 4

Transduction occurs when a phage (i.e. a virus that infects bacteria) infects a bacterial cell (donor) and then transfers some of the bacterial DNA to another bacterial cell (recipient).
If some of this DNA is then incorporated into the recipient cell’s chromosome by homologous recombination, a recombinant cell is formed.

Generalised transduction

• Generalized transduction results from an error in a phage lytic cycle.
• Bacterial genes are randomly transferred from one bacterial cell to another.
• A small piece of the host cell’s degraded DNA could be accidentally packaged within a phage capsid in place of the phage genome during assembly.
• After its release from the lysed host, the phage can attach to another bacterium (the recipient) and inject the piece of bacterial DNA acquired from the first cell (the donor).
• Some of this DNA can subsequently replace the homologous region of the recipient cell’s chromosome, if crossing-over takes place, via homologous recombination, resulting in genetic recombination.

Specialized transduction

• Specialized transduction results from an error in a phage lysogenic cycle.
• Only bacterial genes adjacent to the prophage site are efficiently transferred to another bacterium. (Prophage refers to the phage DNA that is inserted as part of the bacterial genome.)
• An error during the induction of the lytic cycle results in the prophage incorrectly excised from bacterial chromosome and the excised phage DNA incorporated some bacterial genes adjacent to the prophage.
• After its release from the lysed host, the phage can attach to another bacterium (the recipient) and inject the piece of phage DNA carrying bacterial genes from the first cell (the donor) into the recipient bacterium
• Some of this DNA can replace the homologous region of the recipient cell’s DNA by homologous recombination

Question 5

Describe how conjugation (including the role of F plasmids but not Hfr) give rise to variation in prokaryotic genomes

Answer 5

Conjugation involves a direct physical interaction between two bacterial cells and the transfer of genetic material from a donor bacterium to a recipient bacterium.

• The DNA transfer is one-way, i.e. one cell donates DNA, and the other cell receives the DNA.
• The donor uses appendages called sex pili (singular: pilus) to attach to the recipient.
• After contacting a recipient cell, a sex pilus retracts, drawing the donor and recipient cells closer together.
• A temporary cytoplasmic mating bridge then forms between the two cells
• In most cases, the ability to form sex pili and donate DNA during conjugation is due to the presence of an F factor
  –> The F plasmid consists of several genes that are
  required for the production of sex pili and may
  carry genes that confer a growth advantage for
  the bacterium.
• Bacterial cells containing the F plasmid are F+ cells and function as DNA donors during conjugation.
• Bacterial cells lacking the F factor are designated F- cells. These cells function as DNA recipients during conjugation as the F+ condition is transferable.
• Genes within the F factor encode proteins that promote the transfer of one strand of F factor DNA.
• This DNA strand is cut at the origin of transfer, and then the strand travels through the cytoplasmic mating bridge into the recipient cell.
• The other strand remains in the donor cell and is replicated, restoring the F factor DNA to its original double-stranded condition.
• In the recipient cell, the two ends of the newly acquired F factor DNA strand are joined to form a circular molecule, which is then replicated to become double-stranded.
• Each parental strand acts as a template for synthesis of the second strand in its respective cell.

Question 6

Explain how gene expression in prokaryotes can be regulated, through the concept of simple operons (including lac and trp operons), including the role of regulatory genes

Answer 6

In prokaryotes, the cluster of structural genes that encode enzymes of a metabolic pathway under the transcriptional control of a single promoter and operator, in a region on the chromosome, is called an operon.

A typical bacterial operon consists of regulatory sequences (non-coding DNA which includes promoter, operator and terminator) and structural genes. A structural gene is a region of DNA that codes for a protein or RNA molecule that forms part of a structure or has an enzymatic function.

The mRNA that is transcribed is described as a polycistronic mRNA as it contains the coding sequences of two or more structural genes.

Lac Operon:

lacZ gene
- codes for β-galactosidase
- β-galactosidase hydrolyses lactose into glucose and
galactose.
- A side reaction of this enzyme is to convert a
small percentage of lactose into allolactose
lacY gene
- codes for lactose permease,
- lactose permease is a membrane protein required for
transport of lactose into the cell
lacA gene
- codes for galactoside transacetylase

A regulatory gene, lacI gene, lies adjacent to the lac operon. 
- codes for the lac repressor.
- lac repressor is important for the regulation of the lac
operon.
- lacI gene is constitutively expressed at fairly low
levels.
- lacI gene has its own promoter called the i promoter.
It is considered to be a regulatory gene because the
sole function of the repressor protein is to regulate
the expression of structural genes.
- The lacI gene is not considered a part of the lac
operon.
- The lac repressor is synthesised in an
active form and binds to the operator.
- When the lac repressor binds to the operator, the
promoter is blocked from the RNA polymerase, and
transcription of the structural genes is prevented.
- However, the operon is not permanently switched
off as the binding of the repressors to operators is
reversible.
- The ability of the repressor to bind the operator and
inhibit transcription depends on the protein’s
conformation, which is allosterically regulated by an
inducer. Thus, the concentration of the inducer
determines the activity of the operon.

Presence of glucose influences catabolite repression

• cAMP accumulates when concentration of glucose is low. When cAMP accumulates, it binds to CAP.
• This activates CAP and causes it to bind to the CAP site (enhancer).
• Because CAP is an activator, it enhances the rate of transcription of the structural genes.

Trp Operon:

A regulatory gene, trpR gene, located some distance away from the operon.

• codes for the trp repressor.
• trp repressor is important for the regulation of the trp operon.
• trpR gene has its own promoter. It is considered to be a regulatory gene because the sole function of the repressor protein is to regulate the expression of structural genes.
• The trp repressor is synthesised in an inactive form with little affinity for the trp operator.
• Only if tryptophan binds to the trp repressor does the repressor protein change to the active form that can bind to the operator, inhibiting transcription of the structural genes.
• Tryptophan functions as a co-repressor

Question 7

Distinguish between inducible and repressible systems (knowledge of attenuation of trp operon is not required)

Answer 7

Similarities:

1. Both operons consist of a promoter, an operator and a cluster of genes that encode enzymes of a metabolic pathway.
2. Both operon will transcribe their structural genes to form a polycistronic mRNA.

Differences:
Lac vs Trp operon

1. Type of metabolic reaction involved:
   Synthesizes enzymes involved in a catabolic pathway
   vs
   Synthesizes enzymes involved in a anabolic pathway
2. Usual state of the operon
   Usually not expressed/ Turned off
   vs
   Usually expressed/ Turned on
3. Effect of an Effector molecule
   Transcription of the structural genes can be turned on when an inducer (allolactose) binds allosterically to the repressor.
   vs
   Transcription of the structural genes can be turned off when a corepressor (tryptophan) binds to the repressor.
4. Gene product of regulatory gene
   Synthesizes active lac repressor
   vs
   Synthesizes inactive trp repressor
5. Events when lactose/ tryptophan is absent
   Active repressor binds operator, and transcription of structural genes is inhibited.
   vs
   Inactive repressor cannot bind operator, transcription of structural genes occur.
6. Events when lactose/ tryptophan is present
   Allolactose binds to repressor resulting in a conformational change. Inactive repressor cannot bind operator, and transcription is turned on / operon expressed
   vs
   Tryptophan binds repressor resulting in an active repressor. Active repressor binds operator, and transcription is turned off / operon not expressed

Question 8

Describe the Translational Control of prokaryotic genome

Answer 8

Translation Control
(i) Translational repressors
A translational repressor recognises sequences within the mRNA, acting to inhibit translation. These proteins bind to the mRNA to inhibit translation by: ——– Binding near the ribosome-binding site and / or start codon and strategically block the ribosome from initiating translation.
- Binding to the secondary mRNA structures, thereby stabilising these secondary structures, thus preventing initiation of translation by ribosomes.

(ii) Synthesis of antisense RNA Double-stranded RNA can form if a second strand of RNA whose sequence of bases is complementary to the first strand is available. An antisense RNA is an RNA strand that is complementary to a strand of mRNA. It can be synthesised from the non-template (antisense) strand of the double-stranded DNA. When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked. This may occur because:
- the ribosome cannot gain access to the nucleotides in the mRNA or
- antisense RNA-mRNA duplex is quickly degraded by ribonucleases in the cell