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Lecture 7

BCHM 658 Lecture 7: Ph1BiochemTut07-DNARNAProteinsynthesisAnswers2-211211

BCHM - Biochemistry
Course Code
BCHM 658

of 3
Phase 1 Biomolecular Tutorial 7
Protein Synthesis and Antibiotics
Case History
An eleven-year-old girl was admitted to hospital in a critical state. She had been in perfect health until
four days prior to the admission, when fever, abdominal pain, prostration and a mild sore throat had
suddenly developed. On the morning of admission, the child was having difficulty breathing, and a
temperature of 38.9° C (102° F). The tonsils were enlarged bilaterally and covered with a dirty-greyish
green membrane that extended over the entire posterior pharynx, and there was a fetid odour to her
Routine laboratory studies of blood and urine were normal. An electrocardiogram showed abnormality
of the cardiac conducting system and diptheria was therefore suspected.
The patient was given 40,000 units of equine diptheria antitoxin intravenously, and treated with an
antibiotic (erythromycin). The pharyngeal membrane regressed over the next five days, and the patient
was kept in bed for 14 days, by which time the ECG was normal.
1. The antibiotic erythromycin interferes with the process of protein synthesis.
Draw a summary diagram of the various stages from transcription to protein assembly (similar to
that on page 3 of the lecture handouts for Phase 1 lectures L083, L084 & L093) and indicate on
the diagram the stage that erythromycin inhibits .
Erythromycin binds to the larger ribosomal subunit (50S) in prokaryotes, and inhibits the translocation of
ribosome to the next codon on the messenger RNA. This therefore stops protein synthesis at the
elongation stage.
2. Explain why erythromycin inhibits protein synthesis in bacteria, but not in eukaryotic cells and
is therefore useful as an antibiotic.
Bacteria have ribosomes that differ in their size and properties from eukaryotic ribosomes.
Erythromycin binds to the 50S subunit in prokaryotes, but not to the corresponding large ribosomal
subunit in eukaryotic cells (60S). Thus the drug is specific in its action against protein synthesis in
bacteria and has no effect against protein synthesis in the host. (NB mitochondria have a small amount of
their own DNA and the transcription and translation stages are very similar to that in prokaryotes)
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3. For the antibiotics listed below, which all interfere with the overall process of protein synthesis,
indicate their sites of action on the summary diagram.
chloramphenicol: inhibits the peptidyl transferase activity of the 50S ribosomal subunit (elongation stage)
tetracycline: inhibits the attachment of aminoacyl t-RNA to the 30S ribosomal subunit (initiation stage)
rifamycin binds to RNA polymerase and prevents copying of DNA to mRNA (transcription)
puromycin: puromycin resembles part of the structure of an amino acyl t-RNA, thus binds to the large
ribosomal subunit, and causes premature termination of the growing polypeptide chain (termination stage)
4. Indicate which of these antibiotics are specific in their effects on bacterial (prokaryote) protein
All of the drugs are specific inhibitors of prokaryotic transcription and translation, except for puromycin
which affects both prokaryotes and eukaryotes.
5. What effects would an antibiotic that affects prokaryote protein synthesis have on mitochondrial
protein synthesis in the host? Does this affect their clinical uses?
Most of these antibiotics have adverse effects on mitochondrial transcription and translation when
studied in the laboratory. However, in vivo, the effects are minimal, mainly because few drugs cross the
inner mitochondrial membrane, and also because mitochondria have a very low rate of transcription of
their mDNA due to their slow rate of division and replacement. Thus they are OK to use clinically.
6. Summarise 3 ways in which the process of transcription differs between prokaryotes and
Prokaryotes transcription occurs in the cytosol, mRNA not significantly modified,
mRNA may be polycistronic
Eukaryotes transcription occurs in the nucleus, hnRNA greatly modified to become mRNA by
removal of introns, mRNA only codes for one protein
7. Heteronuclear RNA (hnRNA) is modified in several ways in its conversion to mRNA.
Give examples of two of these modifications and explain their functional significance.
Organisation of DNA in eukaryotic genes includes exons and introns (non coding regions). The introns
need to be removed and the exons spliced together to give the correct sequence of triplet codons for the
sequence of amino acids in the final protein.
Capping at the 5’ end with 7-methyl guanine: promotes binding of mRNA to the ribosome, prevents
attack by nucleases
Addition of the polyA tail at the3’ end: aids exit of mRNA through the nuclear pores and confers
stability to mRNA in the cytosol.
8. Briefly summarise the structural differences between tRNA, mRNA and rRNA.
tRNA 75 – 90 nucleotides long, folds to clover leaf 3-D structure, has aa binding site+ anti-codon
mRNA codes for proteins, single stranded, very variable in length, coding region starts with AUG,
rRNA several components of variable size, associate with proteins to form small and large ribosomal
subunits (30S and 50S in prokaryotes = 70S) (40S and 60S in eukaryotes =80S)
9. List the four stages of translation and indicate at which of these stages either ATP or GTP is
Amino acid activation to amino acyl t-RNA (needs ATP)
Formation of initiation complex (needs GTP)
Elongation stage (translocation step needs GTP)
Termination - release of completed polypeptide chain (needs GTP)
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Use the table of the genetic code shown on p 12 of the handout (or in any cell biology text book)
to answer the next 4 questions. Students do not need to remember all the codons by heart, but
they should be able to use the genetic code to relate mutational changes to amino acid changes.
10. Define the term ‘initiation codon’ and state what the triplet codon is.
Start codon, AUG, found at 5’ end of coding region of every mRNA (codes for methionine in eukaryotes
and formyl-methionine in prokaryotes)
11. Define the term stop codon and give one example.
The 3 triplet codons which cause dissociation of the finished polypeptide chain
12. Give one example of an amino acid that has one unique codon.
Methionine (AUG) and also tryptophan (UGG)
13. Give one example of an amino acid that has 6 codons.
leucine arginine serine
14. Name the enzyme responsible for peptide bond formation during the elongation stage of protein
synthesis. State where the enzyme is located and indicate one unusual feature of the enzyme.
Peptidyl transferase, located on the large ribosomal subunit (recent experimental evidence suggests that
its catalytic activity may be due to a region of RNA in the large subunit, not a protein based enzyme).
15 What affect does the toxin produced by the diptheria micro-organism have on protein synthesis,
and why does the patient feel unwell for such a long time?
The toxin produced by the diptheria micro-organism travels in the blood stream to other tissues such as
the heart and kidneys, The toxin covalently modifies the elongation factor required in the translocation
step of protein synthesis, inhibits the synthesis of new proteins and thus causes tissue damage. The toxin
is very stable, and the anti-toxin given by injection only reacts with the free plasma form of the toxin, not
the intra cellular bound form.
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