Pharm Chapter 33: Pharm of Bacterial Infections: DNA Replication, Transcription, and Translation
Three differences between humans and bacteria about DNAproteins, that are targeted by drugs:
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Topoisomerases – regulate supercoiling of DNA and segregation of replicated strands of DNA
RNA polymerases – transcribe DNA to RNA
Ribosomes – translate mRNA into protein
To get all the genetic info to both cells made during cell division, the DNA needs copied, called
replication
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The two resulting copies need segregated, one going to one cell, and the other to the other
In order to express the genes in the DNA, these parts of the DNA are copied onto RNA, called
transcription
mRNA’s are then read to make proteins, called translation
DNA is made of two strands of polymerized deoxyribonucleotides that wind around each other in a
double helix
The 5’ OH of each nucleotide is joined by a phosphate group to the 3’ OH of the next nucleotide, forming
the phosphodiester backbone of each side of the double helix
So double helix is twisted ladder look, the phosphodiester backbone is the sides, and the base pairs are
the rungs of the ladder
The linear sequence of the bases encodes the genetic info
Bacterial chromosomes are usually circular DNA, and eukaryotes usually have linear
During DNA replication, complementary strands of DNA are synthesized bidirectionally, forming two
replication forks
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To initiate this process, the two DNA strands of the double helix must unwind and separate
This forms supercoils, where the polymer overtwists (picture a telephone cord)
o Supercoils increase tension in DNA strands, interfering with further unwinding
o If there were no way to relieve this tension, the entire chromosome would have to
rotate, which would be hard and use lots of energy
Also, when DNA replication is complete, the two progeny copies are wrapped around each other
o In bacteria, since the chromosomes are circular, the intertwined progeny DNA form
interlocking rings called catenanes, that need separated before they can go to their
respective cell
Topoisomerases fix both those problems – they remove excess DNA supercoils during DNA
replication, and separate intertwined progeny DNA
o Topoisomerases do this by breaking, rotating, and re-ligating DNA strands
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Type 1 topoisomerases – form and reseal single-stranded breaks in DNA to decrease
supercoiling
Type 2 topoisomerases – form and reseal single-stranded breaks in both strands to
decrease supercoiling
Both are used during DNA replication to remove excess DNA supercoils
Only type 2 topoisomerases can resolve intertwined copies of double-stranded DNA, to
allow segregation of the DNA to daughter cells
Type 2 topoisomerases are more commonly targeted by drugs
How type 2 topoisomerases work:
 First the enzyme binds a segment of DNA, and forms covalent bonds with
phosphates from each strand, therefore nicking both strands
 Next, it causes a second stretch of DNA from the same molecule to pass through
the break, relieving supercoiling
 This passage of double-stranded DNA through a double-stranded break, is what
allows separation of intertwined copies of DNA following replication
There are two main bacteria type 2 topoisomerases:
 DNA gyrase – can introduce negative supercoils before the DNA strands
separate, and therefore neutralize positive supercoils that form as the strands
unwind
 Topoisomerase 4
Topoisomerases are targets of antibacterial and anticancer drugs
Bacterial transcription:
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Bacterial transcription is catalyzed by RNA polymerase – made of 5 subunits, ơ subunit catalyzes
transcription, and the others catalyze RNA synthesis
Transcription has three stages: initiation, elongation, and termination
Initiation – RNA polymerase separates the strands of a small segment of the double helix DNA,
after its ơ subunit recognized an upstream site
o Once the double helix is unwound to form a single-stranded template, RNA polymerase
initiates RNA synthesis at a start site on the DNA
Elongation- RNA polymerase synthesizes a complementary RNA strand by joining together
ribonucleoside triphsophates via phosphodiester bonds
RNA synthesis proceeds in the 5’3’ direction, until the termination sequence is reached
RNA polymerase is different in bacteria than in humans, so it is a good selective target for drugs
o In bacteria, one RNA polymerase synthesizes all of the RNA in the cell
 Except short RNA primers made by primase for DNA replication
o Bacterial RNA polymerase is made of only 5 subunits
o Eukaryotes have three different RNA polymerases that are all way more complex with
their subunits
Bacterial protein synthesis:
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Once the mRNA transcripts are synthesized, they get translated
The process is similar, but there are many differences between protein synthesis in bacteria and
people
The # and make-up of rRNA’s is different in bacteria ribosomes than in human ribosomes
Bacterial ribosomes are also different enough to be good targets for drugs
o Bacterial ribosomes have a sedimentation coefficient of 70S, and is made of a 30S
subunit and a 50S subunit
o The 30S has a 16S rRNA, while the 50S has both a 23S rRNA and a 5S rRNA
o The rRNA are responsible for the ribosomes key activities, not the proteins
 Includes decoding the mRNA, lining together a.a’s, and translocating the
translating apparatus
o The 70S has two sites to bind tRNA’s:
 The peptidyl (“P”) site – holds the growing peptide chain
 The aminoacyl (“A” aka acceptor) site – holds tRNA that are bringing in new
amino acids to be added
 There’s also an exit (“E”) site that holds tRNA that went through translation,
before they get tossed out the ribosome
Translation has three steps:
o Initiation – the parts of the translation system assemble together
 First the mRNA joins with the 30S subunit of the bacterial ribosome, and with a
tRNA linked to formylated methionine (fMet)
 fMet is the first a.a encoded by every bacterial mRNA
 The tRNA-fMet binds to its start codon (AUG) on the mRNA
 Next, the 50S subunit joins with the 30S subunit to form the complete 70S
ribosome
 At this point, the fMet-tRNA is in the P site of the 70S ribosome
o Elongation – adding amino acids to the carboxyl end of the growing peptide chain, as
the ribosomes moves form the 5’ end to the 3’ end of the mRNA being translated
 Aminoacyl tRNA’s enter the ribosomal A site, and base pair to their
complementary codons on the mRNA
 Use of the correct tRNA requires anticodon recognition between tRNA and
mRNA, and also decoding functions provided by the 16S rRNA in the 30S
ribosomal subunit
 Peptidyl transferase catalyzes the formation of a peptide bond between fMet
and the next amino acid
 So it’s fMet peptide-bonded to the next a.a., which is then linked to the tRNA in
the A site (the tRNA in the A site has accepted the fMet)
 Once the peptide bond has been made, the ribosome advances three
nucleotides further toward the 3’ end of the mRNA
 So the tRNA at the P site moves to the E site, and leaves the ribosome
 The tRNA at the A site that now has both a.a’s, moves to the P site
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 The A site is now open for the new tRNA to come in
 This process is called translocation, where polypeptide chain elongation
happens from multiple cycles of aminoacyl tRNA binding to the A site, peptide
bond making, and translocation
Termination- proteins called release factors recognize the termination codon in the A
site, and activate release of the newly made protein, and dissociation of the ribosome
from the mRNA
The two ribosomal subunits have different jobs
 The 30S – decoding of the mRNA message
 50S – catalyzes peptide bond making
 Translocation uses both of them
The rRNA of the ribosome catalyzes everything and does the work
There are three classes of drugs that target bacterial DNA replication, transcription, and translation:
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Quinolones, rifamycin derivatives, and drugs that target bacterial ribosomes
Quinolones –work in replication
Rifamycin derivatives work in transcription
Many drugs inhibit protein synthesis
o Those that affect the 30S subunit – aminoglycosides, spectinomycin, and tetracyclines
o Those that target the 50s subunit – macrolides, chloramphenicol, lincosamides,
streptgramins, oxazolidinones, and pleuromutilins
Quinolones – bactericidal antibiotics that inhibit bacterial type 2 topoisomerases during DNA replication
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Nalidixic acid –the first quinolone, used in UTIs
New quinolones are fluorinated, called fluoroquinolones – they all end in “oxacin”
o Includes ciprofloxacin, ofloxacin, and levofloxacin
Fluoroquinolones are widely used to treat common respiratory, and GU and GI infections caused
by gram-negative bacteria (ex: E.coli)
Bacteria usually evolve resistance to quinolones through chromosomal mutations in the genes
that encode type 2 topoisomerases, or through changes in expression of membrane porins and
efflux pumps
Quinolones usually don’t cause adverse effects, but could be nausea, vomiting, and diarrhea
Quinolones work by inhibiting one or both of the type 2 topoisomerases in sensitive bacteria
o DNA gyrase and topoisomerase 4
o Quinolones are selective for bacteria, because bacterial topoisomerases are different
than ours structurally
Quinolones usually inhibit DNA gyrase in gram-negatives, and topoisomerase 4 in gram-positives
o One of these gram positives is staph aureus, which has developed resistance to
quinolones
o This is why quinolones are usually used for gram-negatives
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Normally, type 2 topoisomerases bind to and nick both strands of the DNA molecule, allowing
another stretch of the same molecule to pass through the break
o Quinolones inhibit these enzymes before the second segment of DNA can pass
through, keeping it broken
At low doses, quinolones are bacteriostatic (doesn’t kill them, just neutralizes), and inhibit type
2 topoisomerases reversibly
At high doses, quiniolones convert topoisomerases into DNA-damaging agents by stimulating
dissociation of the enzyme from the broken DNA
o Doubly nicked DNA can’t be replicated, and transcription can’t happen either, leading to
cell death
o This is the therapeutic dose, and it’s bactericidal
Rifamycin derivatives – inhibit transcription
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Two synthetic derivatives of the naturally occurring antibiotic rifamycin B, are rifampin
(rifampicin) and rifabutin
The major use of rifampin is in treatment for tuberculosis and other mycobacterial infections
o Rifampin can also be used in meningococcal infections
Rifampin is very effective against mycobacteria that hang out in phagosomes, because rifampin
is bactericidal for both intracellular and extracellular bacteria
Rifamycins inhibit RNA polymerase by forming a stable complex with it
o Both drugs target the β subunit of bacterial RNA polymerase
o Rifampin differs in that it allows initiation of transcription, but then blocks elongation
Rifampin is highly selective for bacteria, so it doesn’t usually cause adverse effects
o May cause rash, fever, nausea, vomiting, and jaundice
Rifampin is often used with antituberculosis drugs due to resistance
Translation inhibitors - target either the 30S or 50S ribosomal subunit of the bacteria
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Translation inhibitors can also affect human ribosomes, causing adverse effects
o One of them, GI issues, is seen in most orally taken antiobiotics, and is due to
eliminating normal gut flora
Also, complete inhibition of bacterial protein synthesis, isn’t enough to kill the bacteria
o They can respond in ways to make them dormant till the treatment is gone
o This makes most translation inhibitors bacteriostatic
Translation inhibitors of the 30S ribosomal subunit:
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Aminoglycosides – they all end in “cin”
o Aminoglycosides are used mainly to treat infections by gram-positive bacteria
o Aminoglycosides need to be taken parenterally (means not by mouth)
o The aminoglycosides include streptomycin, gentamicin, neomycin, kanamycin,
tobramycin, paromomycin, netilmicin, and amikacin
 Streptomycin was the first one discovered
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The ones most used are gentamicin, tobramycin, and amikacin
 They have less toxicity and target more bacteria
Aminoglycosides bind to the 16S rRNA of the 30S bacterial ribosomal subunit
At low concentrations – aminoglycosides induce ribosomes to misread mRNA during
elongation, leading to making of proteins with the wrong amino acids
 So they interfere with mRNA-decoding of the 30S subunit
 Aminoglycosides affect decoding by binding the 30S subunit, causing a shape
change in the 16S rRNA that mimics the shape change that would happen if the
right tRNA anticodon had bound to an mRNA codon
 Then the 30s signals to the 50S to form a peptide bond
At higher concentrations – aminoglycosides completely inhibit protein synthesis
The ribosomes become trapped at the AUG start codon of mRNA
Eventually, accumulation of these abnormal initiation complexes stops translation
Unlike other protein synthesis inhibitors, aminoglycosides are bactericidal
 When drug first enters the cell, it’s poorly transported across bacterial
membranes, creating low concentrations that cause misreads, and making of
proteins with the wrong a.a’s
 Some of these proteins then insert into the membrane and form pores, which
allow aminoglycosides to flood in and stop protein making completely
 So the damage to the membrane can’t be repaired, and things leak out, causing
cell death
Aminoglycosides act synergistically with things that inhibit cell wall making, like βlactams, so the two are often used in combo
 Inhibition of cell wall making increases entry of aminoglycosides into the
bacteria
 This is different than the antagonism that happens between β-lactams and the
bacteriostatic translation inhibitors
Bacteria can become resistant to aminoglycosides three ways:
 Plasmid-encoded making of a transferase enzyme that inactivates
aminoglycosides
 Most important clinically
 Preventing drug entry into the cell
 The 30S ribosomal subunit can mutate
Aminoglycosides can cause the usual general toxicities seen, like hypersensitivity rxns
and drug-induced fever
Aminoglycosides can also cause specific adverse effects of ear and kidney toxicity, and
neuromuscular blockade
 Ear problem is called ototoxicity
 Ototoxicity is the most clinically important problem that restricts use of
aminoglycosides
 Aminoglycosides can accumulate in the ear and damage hair nerves
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Aminoglycosides can also cause acute renal failure by accumulating in the
proximal tubules
The neuromuscular cascade can cause respiratory paralysis
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Spectinomycin
o Also bind to the 16S rRNA of the 30S ribosomal subunit, at a different spot than the
aminoglycosides
o Spectinomycin permits formation of the 70S complex, but inhibits translocation
o Spectinomycin is given parenterally, and is used as an alternative for gonorrhea
Tetrycyclines – all end in “cycline”
o Tetracyclins include chlortetracycline, oxytetracycline, tetracycline, demclcylcine,
methacycline, doxycycline, and minocyclilne
o All of those are similar in structure, and only have minor differences
o Tetracyclines are broad spectrum
o Tetracyclines bind reversiblty to the 16S rRNA of the 30S subunit, and inhibit protein
synthesis by blocking the binding of aminoacyl tRNA to the A site of the ribosome
complex
o This prevents adding more amino acids to the peptide
o Tetracyclines are highly selective though because of their accumulation in bacteria, but
not in human cells
o Tetracyclines enter gram-negative bacteria by passive diffusion through porin proteins
in the outer membrane, followed by active transport across the inner cytoplasmic
membrane
o Tetracyclines enter gram-positive bacteria by active transport
o Humans don’t have the active transport system to take up the tetracyclines
o Resistance to tetrycyclines is developed by forming efflux pumps, or preventing drug
entry
 The ribosomal subunit can also mutate, and the drugs can also be inactivated
o Foods high in calcium, and medicines with divalent and trivalent cations (ex: antacids)
will prevent absorption of tetracyclines, so you usually take tetracyclines on an empty
stomach
o Once in the blood, if tetracylines interact with that same stuff, it will accumulate in bone
and teeth, causing developmental problems, and color changes in the teeth
o Kidney toxicity and GI distress are the two most problematic adverse effects, causing
nausea and vomiting
o Less doxycycline is excreted by the kidney than the other tetracyclines, so they’re the
safer tetracycline to use when there’s kidney problems
 Doxycycline excreted in feces also won’t mess with gut flora, making it more
safe, and less likely to cause nausea, vomiting
Protein synthesis inhibitors targeting the 50S ribosomal subunit
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They bind to the 23S rRNA near the P site
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Macrolides and ketolides
o Macrolides are large lactone rings attached to deoxy sugars
o Erythromycin is the best know macrolide
o Synthetic derivatives of erythromycin are broader in spectrum and better tolerated
 Includes azithromycin and clarithromycin
o Macrolides are very good at treating pulmonary infections, because they penetrate the
lungs well, and can work against intracellular bacteria
o Macrolides are bacteriostatic antibiotics that block translocation in protein synthesis,
by targeting the 23S rRNA of the 50S subunit
 Macrolides block the exit tunnel that the peptide being made goes through
o Resistance to macrolides is usually plasmid-encoded
 Also, esterases can be made to hydrolyze macrolides
o The ribosome binding site can also be mutated, and membranes can be made less
permeable to macrolides, and have pumps to pump out the drug
o In gram positives, resistance is usually do to methylase making
 Methylase modifies the ribosomal target of the macrolides, decreasing its
binding
o Adverse rxns to erythromycin usually involve the GI or liver
 GI is the most common issue, since erythromycin can trigger gut motility, and
cause nausea, vomiting, diarrhea, and sometimes anorexia
 Erythromycin can also cause acute, gall stone hepatitis
o Azithromycin and clarithromycin are usualy better tolerated, but can still cause some
liver issues
o So macrolides like erythromycin, clarithromycin, and azithromycin, are bacteriostatic by
binding the 23S rRNA of the 50S subunit, preventing proteins from exiting the complex
o Telithromycin is a synthetic derivative of erythromycin, but is considered a ketolide, and
not a macrolide
 Works similar to macrolides, but has higher affinity for the 50S ribosomal
subunit, because it can bind another site on the 23S rRNA
 This allows you to use telithromycin on bacteria resistant to macrolides
 Can also cause liver problems
Chloramphenicol - inhibits peptide bond making by preventing tRNA from binding the A site
o Bacteriostatic broad spectrum antibiotic that works against both aerobic and anaerobic
gram-positives and gram-negatives
o Can cause serious toxicity, so that limits its use
o Chloramphenicol is sometimes used for typhoid fever, bacterial meningitis, and
rickettsial diseases, but only when safer alternatives can’t be used
o Chloramphenicol binds the 23S rRNA and inhibits peptide bond formation, by occupying
a site that prevents the aminoacyl tRNA from binding the A site
o Resistance to chloramphenicol include decreased permeability to it and plasmidencoded acetyltransferease
o Chloramphenicol is toxic because it inhibits mitochondrial protein synthesis
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Can cause gray baby syndrome in newborns – they can’t conjugate it to excrete
it, so chloramphenicol accumulates, causing vomiting, hypothermia, gray color,
respiratory problems, and metabolic acidosis
 Chloramphenicol can also cause GI distress, and aplastic anemia
o Chloramphenicol increase the half-life of phytoin and warfarin by inhibiting the P450
enzymes that metabolize them to inactivate them
o Like other bactericiostatic inhibitors of microbe protein making, chloramphenicol
antagonizes the bactericidal effects of penicillin and aminoglycosides
Lincosamides – the major one used is clindamycin
o Clindamycin blocks peptide bond formation
o Clindamycin is used for serious anaerobic infections
o Clindamycin may cause pseudomembranous colitis caused by clostridium difficile
Streptogramins – mix of two things: dalfopristin and quinupristin
o Dalfopristin/quinupristin is used for serious or life-threatening infections cause by
vancomycin-resistant enterococcus or strep
o Dalfopristin/quinupristin inhibit protein making by binding the P site of the 23S rRNA
o Streptogramins dalfopristin/quinupristin are bactericidal against many, but not all
bacteria
Oxazolidinones – like linezolid
o Linezolid works great against drug-resistant gram-positive bacteria
 Includes MRSA, penicillin-resistant strep, and vancomycin-resistant
enterococcus (VRE)
o Linezolid binds the A site to inhibit aminoacyl tRNA binding
Pleuromutilins – like retapamulin
o Retapamulin is a topical treatment for bacterial skin infections
o Retapamulin binds the A site where aminoacyl tRNA would normally bind, and also
bindsteh P site
o Retapamulin will also inhibit peptide bond making
Page 588 – summary of bacterial DNA, RNA , and protein drugs