Bacterial Transcription and Molecular Genetics

Question 1

Where is the energy for bacterial transcription derived from?

Answer 1

The energy is derived from the hydrolysis of the bonds between the phosphates

Question 2

Describe bacterial RNA polymerase.

Answer 2

Bacterial RNA pol is a multi-subunit enzyme composed of:

Core Enzyme:
2 x alpha subunits - enzyme assembly
1 x beta subunit - template binding
1 x beta prime subunit - 5’ to 3’ pol activity

Sigma Unit
1 x sigma unit - enables RNA pol to recognize promoter region

NOTE: Core Enzyme + Sigma unit = Holoenzyme

Question 3

What is the function of the sigma unit in bacterial RNA pol?

Answer 3

Enables the RNA pol to recognize and bind to the promoter region. There a multiple types of sigma subunits and they function to regulate transcription under different conditions.

Question 4

Does transcription require an RNA primer?

Answer 4

No, only DNA replication requires a nucleic acid primer.

Question 5

What is an operon? What does it consist of?

Answer 5

A transcriptional unit containing multiple coding regions under the regulation of a single operator.

Operon Components:

1. Regulatory sequence
2. Promoter region
3. One or more open reading frames (ORFs)

Question 6

What are the 3 steps of Transcription?

Answer 6

1. Initiation
2. Elongation
3. Termination

Question 7

Describe the process of initiation.

Answer 7

1. Holoenzyme binds to the -35 and -10 (Pribnow box) consensus sequences through the sigma subunit
2. DNA around the promoter (Pribnow -10 site) melts forming the transcription bubble

Question 8

Describe the process of elongation.

Answer 8

• Local unwinding continues and creates supercoils
• RNA pol begins to synthesize a transcript from DNA sequence, and several short pieces of RNA are made and discarded
• The elongation phase is said to begin when the transcript exceeds 10 nts in length, at which the sigma unit is released and the core enzyme is able to leave the promoter site.
• During transcription, a short DNA:RNA hybrid helix is formed that helps stabilize the polymerase on the DNA and helps facilitate elongation.
• RNA pol begins polymerization by moving 3’ to 5’ on the DNA template strand, building the complementary RNA strand 5’ to 3’ until it reaches a termination sequence

Question 9

What are the 2 mechanism of termination?

Answer 9

1. Factor-Independent Termination→ Generation of a hairpin in the growing RNA strand that is initiated by a particular sequence in the DNA template strand
   - This sequence allows the RNA to fold back on itself, forming a G-C rich stem plus a loop→ hairpin
   - This hairpin destabilizes the polymerase and terminates transcription
2. Factor-Dependent Termination→This requires the participation of another protein (rho) which a RNA-dependent ATPase which unwinds RNA:DNA duplexes.
   - Rho binds to a C-rich rho recognition site near the 5’ end of the nascent RNA strand and moves along the strand until it reaches the RNA pol paused at the termination site
   - The ATP-dependent helicase reduces the stability of the RNA-DNA duplex behind the polymerase and causes the polymerase to release

Question 10

What are the types of bacterial RNA?

Answer 10

Messenger RNA (mRNA)- very small portion of total RNA in the cell

Transfer RNA (tRNA)- 15-20% of total RNA

Ribosomal RNA (rRNA)- 80% of total RNA

Noncoding RNA (ncRNA)

Question 11

What genes does the lac operon encode?

Answer 11

1. lacZ→ β-galactosidase→ hydrolyzes lactose to galactose and glucose
2. lacY→ permase→ facilitates movement of lactose into the cell
3. lacA→ thiogalactosidase transacetylase→ acetylates lactose

Question 12

What are the components of the lac operon?

Answer 12

1. Operator site→ regulatory region that binds regulatory proteins (Downstream of the promoter site)
2. Promoter site→ binds RNA pol
3. Cap site→ bind regulatory proteins

Question 13

Describe negative regulation in the lac operon.

Answer 13

In the absence of lactose the repressor protein, encoded by lacI, sits on the operator site preventing RNA pol from transcribing the genes.

When lactose (inducer) is present, it binds to the repressor protein and changes the protein’s conformation, causing it to release itself from the operator site and allowing RNA pol to transcribe.

Question 14

Describe positive regulation in the lac operon.

Answer 14

In the absence of glucose, adneylyl cyclase is active which causes levels of cAMP to rise.

cAMP binds to the cAMP-regulatory protein (CRP) can forms the CRP-cAMP complex, that binds to the Cap site, causing RNA-pol to more efficiently initiate transcription .

Question 15

What is catabolite repression in the lac operon?

Answer 15

When both lactose and glucose are present, transcription of the lac operon in negligible because adenylyl cyclase in inhibited in the presence of glucose.
(cAMP = catabolite)

Question 16

What is the difference between a catabolic and anabolic operon?

Answer 16

A catabolic operon directs the synthesis of enzymes responsible for catabolic reactions (break down of substrates)
Example: lac operon (lactose breakdown)

An anabolic operon directs the synthesis of enzymes required for biosynthesis reactions
Example: trp operon (tryptophan biosynthesis)

Question 17

What is a frame shift mutation?

Answer 17

When the entire reading frame is change because of the addition or loss of any number of bases, except for 3.

Question 18

What is a silent mutation?

Answer 18

A mutation that does not change the protein sequence.

Question 19

What is a missense mutation?

Answer 19

A mutation that causes a change in the amino acid encoded by the codon.

Question 20

What is a nonsense mutation?

Answer 20

A mutation that causes the formation of a premature stop codon.

Question 21

What is a deamination mutation?

Answer 21

When the bases of A, C or G are deaminated, resulting in bases that pair differently, causing a change in the sequence during replication.

Question 22

What is an auxotrop?

Answer 22

A mutant that requires nutritional supplementation above the wild type.

Question 23

What is a prototroph?

Answer 23

a phenotype that does not require nutritional supplementation.

Question 24

What does Rifampin/Rifampicin inhibit?

Answer 24

Prokaryotic RNA pol

Question 25

What is an Ames Test?

Answer 25

It is a rapid test carried out in bacteria that allows you to test chemicals for their effect on DNA. In an Ames test, a particular enzyme required for histidine synthesis is inactivated, and a mutation in the bacterial’s DNA is required to make this enzyme work.

Colony formation = mutagenic chemical

Question 26

What is a transposable element and how does it work?

Answer 26

Transposable elements are made of a transposon and transposase.

1. Transposons (Tn)- mobile segments of DNA that move in an essentially random manner from one site to another on the same or a different chromosome
   - This movement in mediated by transposase, which is an enzyme that is encoded by the Tn itself
2. Transposase- a specialized recombinase that recognizes transposon DNA and copies it to other locations in the genome

• Transposable elements are selected for in bacterial populations and are medically important because they can be associated with drug resistance
• They can also move among different plasmids and genomes
• The growing problem of antibiotic-resistant bacteria is a consequence os the exchange of plasmids among bacterial cells. If the plasmid contains Tns carry antibiotic resistance genes, the recipient bacteria gain resistance to one or more antimicrobial drugs)

Question 27

What is homologous recombination?

Answer 27

Recombination between 2 DNAs that have similar sequences.

Question 28

What is transformation?

Answer 28

The uptake of naked DNA by bacteria. Bacteria that can take up DNA are said to by “competent”.

External dsDNA binds to DNA binding proteins on the cell surface and nucleases degrade the DNA so that a single strand of DNA is internalized by the cell. The single strand of DNA recombines with homologous regions in the bacterial chromosome, creating a transformed bacterial cell.

NOTE: The ability of bacteria to take up DNA from other bacteria (that have released their DNA into the surrounding environment) means that they can readily exchange plasmid and genomic DNA in the gut. This is a mechanism of transmissible drug resistance.

Question 29

What are conjugation plasmids and how do they work?

Answer 29

Conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. Conjugation plasmids are self-transmissible or mobilizable. They encode a structure called a pilus, which mediates contact with other bacteria and allows transfer of plasmid to recipient bacteria.

Another means of transferring antibiotic resistant genes.

Question 30

What is a conjugative transposon?

Answer 30

Combines the properties of transposons with conjugation plasmids. They are integrated in the bacterial chromosome, but they can excise and transfer from the chromosome of the donor to the chromosome of the recipient or into plasmids.

NOTE: Can transfer between gram negative and gram positive cells.

Question 31

What kind of material can a bacteriophage carry?

Answer 31

RNA or DNA, single or double stranded.

Question 32

What is transduction?

Answer 32

Transduction is the ability of lysogenic phage to acquire bits of the host genome and transfer the DNA to a new host.

Question 33

How do bacteria protect themselves from incoming phage and foreign DNA?

Answer 33

Bacteria have evolved restriction systems which cleave DNA at specific sequences which do not occur or are methylated (protected) in bacterial DNA. This is process is achieved by restriction enzymes, which are homodimers that bind and cleave DNA at sites which are palindromic or have 2-fold symmetry.