Antibiotics + DNA Flashcards
What are antibiotics?
Major targets?
Disruptors of bacterial structures and functions.
Bacteriostatic = Inhibits growth and reproduction, but doesn’t directly kill.
Bacteriocidal - directly kill bacteria.
Cell-wall synthesis
DNA gyrases
Protein synthesis - 30s or 50s inhibition, tRNA inhibition,
DNA/RNA polymerases.
What are major targets for antibiotics?
Examples:
Cell-wall synthesis:
Penicillins, Beta-lactams, Carbapenems.
DNA gyrase:
Quinolones/Fluoroquinolones.
Protein synthesis - 30s inhibition:
Tetracyclin, streptomycin.
or 50s inhibition:
Erythromycin, Chloramphenicol.
tRNA inhibition:
Puromycin.
DNA/RNA polymerases:
Rifampines
Actinomycin = RNA elongation.
How can you inhibit nucleic acid synthesis?
Quinolones/Fluoroquinolones:
Inhibit DNA polymerisation by antagonising Gyr A subunit of DNA gyrase.
DNA gyrase is a Type II topoisomerase - Heterotetramer.
GyrA subunits involved in cutting and rejoining DNA.
DNA gyrase vital to relax positive supercoils that accumulate ahead of the replication fork in DNA replication.
= Vital for DNA replication.
Facilitates chromosomal condensation by forming negative supercoils.
= Without DNA gyrase, unable to effectively manage DNA topology and will lead to DNA breaks and failure to replicate DNA,
Eukaryotes have other Type II topoisomerases, not DNA gyrase.
How does transcription occur in bacteria/archaea?
Within cytoplasm, since no nucleus.
Promotor region binds RNA polymerase.
Start codon to Stop codon - neither translated.
There are no introns in bacterial genes, only a few in archaeal genes.
Most genes are structured as operons.
-Permitting concerted production of n proteins with related functions.
E.G lactose operon.
Genes are read in the 3’ to 5’ direction by RNA polymerase, because this ensures the mRNA can be read in 5’ to 3’ for translation.
RNA polymerase adds only to 3’ OH group of RNA strand.
How does prokaryotic translation occur?
In what direction are DNA and RNA read?
There is cytoplasmic coupling:
Transcription via numerous RNA polymerases simultaenously transcribing a single gene, with each mRNA translated by many ribosomes at different points = Polyribosome.
Genes are read in the 3’ to 5’ direction by RNA polymerase, because this ensures the mRNA can be read in 5’ to 3’ for translation.
RNA polymerase adds only to 3’ OH group of RNA strand.
mRNA read in 5’ to 3’ direction.
mRNA is polycistronic - A single long strand of mRNA will provide template for multiple different proteins (Operon)
What is the structure of RNA polymerase in bacteria?
There is the core Enzyme, made of 5 subunits.
= Tetrameric.
Beta subunit with catalytic site= polymerase activity.
Beta’ involved in coordinating Mg2+ ions.
The core enzyme can function on its own, but requires a sigma factor to transcribe at the right site.
Sigma factor required for promotor region binding.
= When sigma factor bound, it becomes a HOLOENZYME.
7 sigma factors, correspond to 7 promoters, so there are 7 different holoenzymes.
(In E.Coli)
What is the structure and arrangement of bacterial promotor regions?
Bacterial promoter regions are rich in A and T nucleotide bases.
There are conserved sequences in -35 and -10 positions.
The promoter sequence is recognised by the sigma factor of RNA polymerase Holoenzyme.
When transcription initiated, sigma factor is released.
no. of sigma factors differes by species, but 7 in E.Coli.
= Allow transcription of gene sets, for heat shock response.
How do RNA polymases differ between bacteria, archaea and eukaryotes?
Bacteria have only 1 Enzyme.
Archaea only have 1 enzyme.
Eukaryotes have 3 different enzymes in total.
There are 5 subunits with the sigma factor added in bacteria.
Archaea can have between 10-15 subunits.
Eukaryotes have 10-16 subunits.
RNA polymerase similarity between eukaryotes and archaea suggest closer evolutionary relationship than with bacteria.
How can RNA polymerase be inhibited?
Rifampicin - inhibits transcription by interacting with catalytic Beta subunit of RNA polymerase, preventing DNA transcription.
ACtinomycin - Binds next to gene promoter region, inhibiting the binding of the RNA holoenzyme (RNA pol and sigma factor).
Both BACTERICIDAL
How are ribosomes structured in bacteria and archaea?
Bacteria and Archaea have 70s Ribosomes containing:
Small ribosomal subunit is 30S.
30s contains 16s rRNA and proteins.
Large ribosomal subunit is 50S.
50S contains 23s rRNA and 5s rRNA, with proteins.
How can ribosomes be inhibited in bacteria and archaea?
70s ribosomes can be inhibited by:
Chloramphenicol in bacteria.
Chloramphenicol binds to 50s Large ribosomal subunit, blocking action of peptidyl transferase and stops amino-acyl tRNA from entering active site.
Anisomycin in Archaea.
How to block protein synthesis in bacteria?
Inhibitors of protein synthesis are bacteriostatic.
Chloramphenicol binds to 50s large ribosomal subunit in Site A - inhibiting binding of amino-acyl tRNA (Charged tRNA) from binding to A site of ribosome.
Macrolides - reversible binding to P site of 50s large ribosomal subunit, to prevent transpeptidation and translocation.
Tetracyclins - reversibly bind to 30S subunit, prevent amino-acyl tRNA attaching to A site.
Aminosides - bind to 30S subunit, at therapeutic conc. prevent elongation of peptide chain by blocking initiating complex.
What is the tree of life by Carl Woese?
Supporting arguments?
Common ancestor diverges to form bacteria and archaea, with archaea more closely related to eukaryotes.
Amino-acyl tRNA to initiate translation in bacteria is formylMet tRNA, but in archaea and eukaryotes is Met-tRNA.
Eukaryotes and Archaea are sensitive to Anisomycin to inhibit 50S subunit, whereas bacteria are not, only to chloramphenicol.
Archaea and Eukaryotes require TATA binding proteins (TBPs) to bind RNA pol complex to DNA… In bacteria, it requires sigma factor.
No. of subunits is 10-15, 10-16 in archaea vs eukaryotes, and 5 including sigma factor in bacteria.
Cytoskeletal polymer regulating proteins - lacking in bacteria, Porifilins in archaea and eukaryotes.
= with homologous structures.
Crenactin in Archaea is more homologous to Eukaryotic Actin, than MreB in bacteria.
= Archaeal cytoskeleton is a intermediate between bacterial and eukaryotic… since contains homologs to both, but adds profilins.
What is the process of translation?
THere is active acylation of tRNA with amino acids.
= Forming cognate amino-acyl tRNA.
Anti-codon sequence of cognate tRNA recognises and binds to specific codon on mRNA within A site.
Peptidyl transferase catalyses addition of AA to growing peptide chain during translocation to P site.
With peptide growing uo through the ribosomal exit tunnel.
When translocated to P site, tRNA is released.
Upon arrival of stop codon, there is ribosomal dissociation and dissassembly into its 2 subunits.
What are Operons?
50% of bacterial genes are contained within operons.
A section of DNA in bacterial genomes containing numerous genes, but transcribed as a single, polycistronic mRNA
Genes encoding for different proteins but which are involved in similar functions/metabolic process.
Lactose operons.
