8.2 - Antibiotics

Question 1

What are antibiotics originally?

Answer 1

Secondary metabolites produced by bacteria to survive in their environment.

Question 2

Explain an important antibiotic experiment?

Answer 2

(1) RNA molecules were labeled with 3-H radio-label uridine.
(2) Proteins were labeled with 14-C radio-label Amino acids.
(3) This labeling will allow us to track the amount of RNA and protein in E.coli.
(4) It was observed that normally, under good conditions, RNA and protein concentration increases in E. coli over time.
(5) When Rifampicin antibiotic was added at t = 0 with the 3-H and 14-C radio labels the protein increased then plateaud whilst the RNA increased then rapidly fell and then plateaued

Question 3

What is the reason for rapid decline in RNA with antibiotic addition?

Answer 3

The rapid decline in RNA is due to the fact that the mRNA is being used to translate proteins and no new mRNA is generated. The plateau in RNA represents the stable RNA molecules that will still be there (rRNA, tRNA). Although there is no formation of new RNA, the previously formed stable RNAs remain. The decrease in RNA is mostly unstable RNA (i.e. mRNA).

Question 4

Why does protein concentration just plateau with antibiotic addition?

Answer 4

No new proteins are made once the antibiotic has been added, as no new RNAs are formed. However, the previously formed proteins remain in the cell. The proteins are stable

Question 5

What is rifampicin?

Answer 5

A secondary metabolite of Steptomyces

Question 6

How does rifampicin work?

Answer 6

Rif binds the RNA channel in the RNA polymerase. This is the channel from which the newly transcribed RNA molecule exists. It specifically binds parts of the beta subunit.

Question 7

What does rifampicin block?

Answer 7

Binding will allow ongoing transcription but it will block new rounds of transcription. In other words, Rif blocks the initiation of transcription.

Question 8

Why is rif a broad spectrum antibiotic?

Answer 8

Because bacteria have very similar RNA polymerases

Question 9

What are the two ways rif resistance can arise?

Answer 9

(1) Resistance to rifampicin arises from mutations that alter the Rif binding site on the RNA polymerase resulting in decrease affinity for rifampicin.These mutations are in the rpoB gene, which encodes the beta subunit of the RNA polymerase.
(2) Resistance could also arise through mutations in the amino acid sequence of the channel.However, this also means that there is a lower affinity of RNA movement through the channel.

Question 10

What does streptomycin inhibit?

Answer 10

Protein synthesis initiation

Question 11

How does streptomycin work?

Answer 11

It binds to the 16S rRNA of the small ribosomal subunit (30S subunit) of bacterial ribosomes. This binding interferes with the binding of the formyl-methionyl (fMet) tRNA to the 30S subunit. If fMet tRNA can’t bind to the 30S subunit, protein synthesis cannot occur

Question 12

What is fMet and fMet tRNA?

Answer 12

fMet is the starting residue in bacterial protein synthesis. The fMet tRNA recognizes the 5’ AUG 3’ start codon. fMet is thus encoded by the same codon as methionine

Question 13

How may streptomycin resistance arise?

Answer 13

Mutations in the binding site of streptomycin on 30S subunit are associated to antibiotic resistance

Question 14

What does Tetracycline inhibit?

Answer 14

protein synthesis initiation

Question 15

How does Tetracycline work?

Answer 15

By binding and blocking the A site of the 30S subunit of bacterial ribosomes. So Tetracycline inhibits the attachment of aminoacyl tRNAs to the A site

Question 16

How might Tetracycline resistance arise?

Answer 16

Antibiotic resistance involves gene that encodes for efflux pumps or ribosomal protection proteins.

Question 17

What do efflux pumps do?

Answer 17

They actively eject tetracycline from the cell.

Question 18

How do ribosomal protection proteins work?

Answer 18

Ribosomal protection proteins interact with the ribosomes and remove tetracycline from it, allowing translation to occur.

Question 19

Why are mammalian cells less vulnerable to the effect of tetracycline?

Answer 19

Because bacteria actively pump tetracycline into their cytoplasm, even against a concentration gradient, whereas mammalian cells do not.

Question 20

What does Chloramphenicol inhibit?

Answer 20

Protein synthesis elongation

Question 21

How does Chloramphenicol work?

Answer 21

It binds to residues on the 23S rRNA of the 50S ribosomal subunit and inhibiting peptidyl transferase activity of the bacterial ribosome. The peptidyl transferase activity consists of forming a peptide bond between 2 adjacent amino acids.

Question 22

What are the three mechanisms of antibiotic resistance in Chloramphenicol?

Answer 22

(1) Reduced membrane permeability to the antibiotic.
(2) Mutation of the antibiotic binding site on the 50S ribosomal subunit.
(3) Encoding a chloramphenicol acetyl transferase.
o This enzyme is encoded by the cat-gene.
o It inactivates chloramphenicol by covalently linking it to one or two acetyl groups.

Question 23

What does Erythromycin inhibit?

Answer 23

Protein synthesis elongation

Question 24

How does Erythromycin work?

Answer 24

It binds to the 50S bacterial ribosomal subunit. It inhibits the transfer of the tRNA from the A site to the P site. The A site will therefore remain occupied and therefore, the addition of an incoming tRNA is inhibited.

Question 25

What are the three mechanisms of antibiotic resistance in erythromycin?

Answer 25

• Target site alteration
• Antibiotic modification
• Altered antibiotic transport